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Genome-wide RNA-mediated interference screen identifies miR-19 targets in Notch-induced T-cell acute lymphoblastic leukaemia

Abstract

MicroRNAs (miRNAs) have emerged as novel cancer genes. In particular, the miR-17–92 cluster, containing six individual miRNAs, is highly expressed in haematopoietic cancers and promotes lymphomagenesis in vivo. Clinical use of these findings hinges on isolating the oncogenic activity within the 17–92 cluster and defining its relevant target genes. Here we show that miR-19 is sufficient to promote leukaemogenesis in Notch1-induced T-cell acute lymphoblastic leukaemia (T-ALL) in vivo. In concord with the pathogenic importance of this interaction in T-ALL, we report a novel translocation that targets the 17–92 cluster and coincides with a second rearrangement that activates Notch1. To identify the miR-19 targets responsible for its oncogenic action, we conducted a large-scale short hairpin RNA screen for genes whose knockdown can phenocopy miR-19. Strikingly, the results of this screen were enriched for miR-19 target genes, and include Bim (Bcl2L11), AMP-activated kinase (Prkaa1) and the phosphatases Pten and PP2A (Ppp2r5e). Hence, an unbiased, functional genomics approach reveals a coordinate clampdown on several regulators of phosphatidylinositol-3-OH kinase-related survival signals by the leukaemogenic miR-19.

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Figure 1: miR-19 enhances cytokine independent survival in vitro.
Figure 2: Pooled miRNA screen for tumorigenic activities within the 17–92 cluster and its paralogues.
Figure 3: miR-19 is a novel T-ALL oncogene.
Figure 4: Gene expression analysis of parental and miR-19-transduced FL5-12 cells.
Figure 5: Genetic screen for shRNAs that phenocopy miR-19 in lymphocyte survival.
Figure 6: Summary of the screen result.
Figure 7: The identified genes are actual targets of miR-19.
Figure 8: miR-19 acts through multiple negative regulators of PI(3)K-related survival signals.

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Acknowledgements

We thank Lin He, S.W. Lowe and G. Hannon for access to shRNA and miRNA screening technologies; W. Pear, J. Huse and E.C. Holland for plasmids; H. Zhu for technical assistance; the Memorial Sloan-Kettering (MSK) animal facility and Research Animal Resource Center (RARC), A. Viale of the MSK Genomics Core, H. Zhao of the cBIO program, and J. Schatz and J. Massagué for editorial advice; and V. Murty for cytogenetic and fluorescence in situ hybridization analyses. This work was supported by grants from the American Cancer Society, the Geoffrey Beene Cancer Center, the Leukemia Research Foundation, the Louis V. Gerstner Foundation, the May and Samuel Rudin Foundation, the NY Community Trust and the William and Blanche Foundation (to H.-G.W.), from the Andrew Seligson Memorial Clinical Fellowship (to J.Z.), and NYStar (to P.J.P.). A.F. is supported by R01CA120196, the WOLF Foundation, the Rally across America Foundation and the Leukemia and Lymphoma Society (grants 1287-08 and 6237-08), and A.F. is a Leukemia & Lymphoma Society Scholar.

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K.J.M, A.L.W. and E.O. performed experimental design and analysis. K.d.K., T.P. and A.F. conducted T-ALL translocation analysis. K.M., J.Z., T.J. and K.C. performed the screen and analysis. A.A.K., C.S.L. and J.S.P. did data analysis. P.J.P. generated the shRNA library. W.T. was responsible for clinical specimens. H.-G.W. designed the study and wrote the paper.

Corresponding author

Correspondence to Hans-Guido Wendel.

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The authors declare no competing financial interests.

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Mavrakis, K., Wolfe, A., Oricchio, E. et al. Genome-wide RNA-mediated interference screen identifies miR-19 targets in Notch-induced T-cell acute lymphoblastic leukaemia. Nat Cell Biol 12, 372–379 (2010). https://doi.org/10.1038/ncb2037

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