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In vivo RNAi screening identifies regulators of actin dynamics as key determinants of lymphoma progression

Abstract

Mouse models have markedly improved our understanding of cancer development and tumor biology. However, these models have shown limited efficacy as tractable systems for unbiased genetic experimentation. Here, we report the adaptation of loss-of-function screening to mouse models of cancer. Specifically, we have been able to introduce a library of shRNAs into individual mice using transplantable Eμ-myc lymphoma cells. This approach has allowed us to screen nearly 1,000 genetic alterations in the context of a single tumor-bearing mouse. These experiments have identified a central role for regulators of actin dynamics and cell motility in lymphoma cell homeostasis in vivo. Validation experiments confirmed that these proteins represent bona fide lymphoma drug targets. Additionally, suppression of two of these targets, Rac2 and twinfilin, potentiated the action of the front-line chemotherapeutic vincristine, suggesting a critical relationship between cell motility and tumor relapse in hematopoietic malignancies.

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Figure 1: In vivo RNAi screening strategy.
Figure 2: Functional validation of shRNAs targeting putative cell-motility genes.
Figure 3: Rac2 suppression impairs lymphoma cell migration and extends animal survival.
Figure 4: Suppression of Rac activity delays disease progression and potentiates the action of the chemotherapeutic vincristine.

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Acknowledgements

We would like to thank E. van Veen for help with motility assays, C. Whittaker and J. Pritchard for bioinformatic processing of sequencing data, H. Thompson for expert technical assistance, and members of the Hemann laboratory for helpful advice and discussions. M.T.H. is a Rita Allen Fellow and the Latham Family Career Development Assistant Professor of Biology and is supported by US National Institutes of Health RO1 CA128803-01. C.E.M. is supported by the MIT Department of Biology training grant, and E.E.H. and E.D. were supported by MIT's UROP program. Additional funding was provided by the Integrated Cancer Biology Program grant 1-U54-CA112967 to F.B.G. and M.T.H.

Author information

Authors and Affiliations

Authors

Contributions

C.E.M. and M.T.H. designed the experiments and wrote the manuscript. C.E.M. and E.E.H. performed the screen and subsequent validation experiments. E.D. and M.T.H. performed initial GFP dilution experiments. F.B.G. designed motility experiments.

Corresponding author

Correspondence to Michael T Hemann.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1 and 2 (PDF 1513 kb)

Supplementary Video 1

Suppression of Rac2 causes motility defects in lymphoma cells. Lymphoma cells were infected with a vector coexpressing a Rac2 shRNA and GFP (green), or a vector control that expresses the fluorescent protein Cherry (red). These cell populations were mixed and plated on fibronectin-coated plates, stimulated with SDF (100 ng/mL), and imaged for approximately 30 minutes. Representative cells are shown. In each movie, two unstained apoptotic (black) cells are also shown. (MOV 4876 kb)

Supplementary Video 2

Suppression of Rac2 causes motility defects in lymphoma cells. Lymphoma cells were infected with a vector coexpressing a Rac2 shRNA and GFP (green), or a vector control that expresses the fluorescent protein Cherry (red). These cell populations were mixed and plated on fibronectin-coated plates, stimulated with SDF (100 ng/mL), and imaged for approximately 30 minutes. Representative cells are shown. In each movie, two unstained apoptotic (black) cells are also shown. (MOV 4876 kb)

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Meacham, C., Ho, E., Dubrovsky, E. et al. In vivo RNAi screening identifies regulators of actin dynamics as key determinants of lymphoma progression. Nat Genet 41, 1133–1137 (2009). https://doi.org/10.1038/ng.451

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