RNA-binding protein IGF2BP3 targeting of oncogenic transcripts promotes hematopoietic progenitor proliferation
Jayanth Kumar Palanichamy, … , Jeremy R. Sanford, Dinesh S. Rao
Jayanth Kumar Palanichamy, … , Jeremy R. Sanford, Dinesh S. Rao
Published March 14, 2016
Citation Information: J Clin Invest. 2016;126(4):1495-1511. https://doi.org/10.1172/JCI80046.
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Research Article Hematology

RNA-binding protein IGF2BP3 targeting of oncogenic transcripts promotes hematopoietic progenitor proliferation

Abstract

Posttranscriptional control of gene expression is important for defining both normal and pathological cellular phenotypes. In vitro, RNA-binding proteins (RBPs) have recently been shown to play important roles in posttranscriptional regulation; however, the contribution of RBPs to cell specification is not well understood. Here, we determined that the RBP insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is specifically overexpressed in mixed lineage leukemia–rearranged (MLL-rearranged) B-acute lymphoblastic leukemia (B-ALL), which constitutes a subtype of this malignancy associated with poor prognosis and high risk of relapse. IGF2BP3 was required for the survival of B-ALL cell lines, as knockdown led to decreased proliferation and increased apoptosis. Enforced expression of IGF2BP3 provided murine BM cells with a strong survival advantage, led to proliferation of hematopoietic stem and progenitor cells, and skewed hematopoietic development to the B cell/myeloid lineage. Cross-link immunoprecipitation and high throughput sequencing uncovered the IGF2BP3-regulated transcriptome, which includes oncogenes MYC and CDK6 as direct targets. IGF2BP3 regulated transcripts via targeting elements within 3′ untranslated regions (3′UTR), and enforced IGF2BP3 expression in mice resulted in enhanced expression of Myc and Cdk6 in BM. Together, our data suggest that IGF2BP3-mediated targeting of oncogenic transcripts may represent a critical pathogenetic mechanism in MLL-rearranged B-ALL and support IGF2BP3 and its cognate RNA-binding partners as potential therapeutic targets in this disease.

Authors

Jayanth Kumar Palanichamy, Tiffany M. Tran, Jonathan M. Howard, Jorge R. Contreras, Thilini R. Fernando, Timothy Sterne-Weiler, Sol Katzman, Masoud Toloue, Weihong Yan, Giuseppe Basso, Martina Pigazzi, Jeremy R. Sanford, Dinesh S. Rao

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Figure 2

IGF2BP3 knockdown leads to disruptions of cell growth and increased apoptosis.

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IGF2BP3 knockdown leads to disruptions of cell growth and increased apop...
(A) IGF2BP3 expression in human B-ALL cell lines. (B) Schematic of lentiviral vector used for IGF2BP3 knockdown. (C) IGF2BP3 knockdown, measured by qPCR shown in RS4;11 cell line (t test; ***P = 0.0005). (D) Cell cycle analysis with propidium iodide staining. (E) MTS assay showing significantly reduced cell proliferation with IGF2BP3 knockdown. (F) Western blot showing IGF2BP3 expression after CRISPR-Cas9–mediated targeting using the Cr1 or Cr2 constructs. Cr1-mediated targeting results in some residual protein. β-Actin is used as a loading control. (G) MTS assay showing significantly reduced cell proliferation after Cr2 targeting (t test; **P ≤ 0.01 for all marked comparisons). (H) Cell cycle analysis by propidium iodide staining showing increased cell death (sub-G1 peak) in Cr2-expressing cells. (I) Increased annexin V staining in Cr2-targeted cells with IGF2BP3 KO. I3, IGF2BP3. Experiments were conducted 3× for validation. Data represent mean ±SD. See also Supplemental Figure 2. UbC, ubiquitin C promoter; Puro, puromycin; LC, lentiCRISPR control.