Abstract
As the most abundant internal RNA modification in eukaryotes, N6-methyladenosine (m6A) modification has been recently linked to various physiological and pathological processes, including tumorigenesis. m6A marks on mRNAs are mainly deposited by the METTL3/METTL14/WTAP methyltransferase ("writer") complex and could be removed by "eraser" proteins such as FTO or ALKBH5. We have shown recently that METTL14 (Weng H et al., Cell Stem Cell, 2018) and FTO (Li Z et al., Cancer Cell, 2017) both function as oncogenes in acute myeloid leukemia (AML), which together with the finding that METTL3 is also oncogenic in AML suggest that the m6A machinery plays critical roles in AML pathogenesis.
Similar to other epigenetic modifications, the effect of m6A modification relies on effector proteins, the so-called m6A "reader" proteins. We recently identified a new class of m6A reader proteins, the IGF2BP protein family, which stabilize m6A-containing mRNAs and promote their translation through their K Homology (KH) domains (Huang H et al., Nature Cell Biology, 2018). Despite the well-studied oncogenic roles of IGF2BPs in solid cancer, little is known on the functions of IGF2BP proteins in leukemia.
By analyzing expression of the IGF2BPs in AML using The Cancer Genome Atlas (TCGA) dataset, we found that one of the three IGF2BP genes, IGF2BP2, is highly expressed in AML compared to the vast majority of other types of cancer. Our qPCR assays also showed a significant elevated expression of IGF2BP2 in mononuclear cells from primary AML patients with various types of chromosome translocation (t(11q23), n=17, P<0.0001; t(15;17), n=5, P=0.0021; t(8;21), n=7, P<0.0001; inv(16), n=6, P<0.0001) compared to those from healthy donors (n=15). Knockdown of IGF2BP2 inhibits Mll-AF9-mediated colony forming in vitro (sh#1 vs. shCtrl, P=0.0113; sh#2 vs. shCtrl, P=0.0136), whereas overexpression of wild-type IGF2BP2 (wt vs. vehicle, P=0.0025), but not the inactive KH3-4-mutated form (mut vs. vehicle, P=0.3178), promotes cell immortalization. In addition, IGF2BP2-promoted cell transforming ability of Mll-AF9 was largely abolished in the absence of Mettl14 in bone marrow cells from Mettl14 conditional knockout mice (IGF2BP2/Ctrl in the presence of Mettl14=1.90±0.15, P=0.0030; IGF2BP2/Ctrl in the absence of Mettl14=1.28±0.10, P=0.0323). Such data indicate that the effect of IGF2BP2 on promoting cell immortalization is m6A dependent. Moreover, depletion of Igf2bp2 delays in vivo leukemogenesis mediated by Mll-AF9 and greatly prolonged the survival of recipient mice (shCtrl, medium survival=81 days, n=7; shIgf2bp2, medium survival>200 days, n=7; P=0.0031).
To better understand the molecular mechanism underlying the role of IGF2BP2 in AML, we performed RNA-seq in IGF2BP2 knockdown AML cells or control cells. Integrated analysis of the RNA-seq data with our previously published cross-linking immunoprecipitation (CLIP) data (GSE90639 and GSE21918) reveals that targets of IGF2BP2 are enriched in biosynthesis of amino acids (P=0.0079) and metabolic pathways (P=0.0160). Expression changes of selected target genes, such as MYC, PHGDH, and ASNS, were confirmed by qPCR and immunoblotting in IGF2BP2 knockdown cells, with similar pattern being observed in METTL3 or METTL14 depleted cells. PHGDH encodes the key metabolic enzyme that catalyzes the rate-limiting step of the serine (Ser) biosynthesis pathway, whereas ASNS encodes an enzyme that converts aspartate (Asp) and glutamine (Gln) to asparagine (ASN) and glutamate (Glu). Metabolic profiling reveals that the synthesis of selected amino acids, including Ser, Asn, and Glu, was decreased in IGF2BP2-depleted cells (P=0.0415 for Ser, P=0.0109 for Asn, P=0.0018 for Glu), similar to that in METTL3 (P=0.0433 for Ser, P=0.0155 for Asn, P=0.0005 for Glu) or METTL14 (P=0.0478 for Ser, P=0.0057 for Asn, P=0.0043 for Glu) depleted cells. As expected, shRNA-mediated knockdown of IGF2BP2 in AML cells resulted in a significant inhibition on the glycolytic rate (P=0.0029 for sh#1, P=0.0024 for sh#2) and cell growth (P<0.0001 for both shRNAs from Day 1 to Day 4). Collectively, our data demonstrate a critical role of IGF2BP2 in AML pathogenesis as an m6A reader and provide novel evidence on the involvement of the m6A machinery on regulation of cell metabolism, which may have therapeutic implications for AML.
Disclosures
No relevant conflicts of interest to declare.
Integrated analysis reveals distinct molecular, clinical, and immunological features of
B7‐H3
in acute myeloid leukemia