Mutations in RABL3 alter KRAS prenylation and are associated with hereditary pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma is an aggressive cancer with limited treatment options1. Approximately 10% of cases exhibit familial predisposition, but causative genes are not known in most families2. We perform whole-genome sequence analysis in a family with multiple cases of pancreatic ductal adenocarcinoma and identify a germline truncating mutation in the member of the RAS oncogene family-like 3 (RABL3) gene. Heterozygous rabl3 mutant zebrafish show increased susceptibility to cancer formation. Transcriptomic and mass spectrometry approaches implicate RABL3 in RAS pathway regulation and identify an interaction with RAP1GDS1 (SmgGDS), a chaperone regulating prenylation of RAS GTPases3. Indeed, the truncated mutant RABL3 protein accelerates KRAS prenylation and requires RAS proteins to promote cell proliferation. Finally, evidence in patient cohorts with developmental disorders implicates germline RABL3 mutations in RASopathy syndromes. Our studies identify RABL3 mutations as a target for genetic testing in cancer families and uncover a mechanism for dysregulated RAS activity in development and cancer.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Whole-genome sequencing in a family cluster of pancreatic cancer identifies a germline nonsense mutation in RABL3.
Fig. 2: RABL3 mutation promotes cell proliferation in vitro and cancer in zebrafish.
Fig. 3: RABL3 mutation dysregulates KRAS activity.
Fig. 4: Homozygous rabl3-TR mutants resemble human RASopathy syndromes.

Data availability

RNA-seq data are available through GEO under accession GSE129081. Interacting proteomic data are available through Peptide Atlas under accession PASS01355. Additional data generated in this study are available within the paper and in the supplementary information.

References

  1. 1.

    Howlader, N. et al. SEER cancer statistics review, 1975-2011. National Cancer Institute http://seer.cancer.gov/csr/1975_2011/ (2014).

  2. 2.

    Rustgi, A. K. Familial pancreatic cancer: genetic advances. Genes Dev. 28, 1–7 (2014).

    CAS  Article  Google Scholar 

  3. 3.

    Williams, C. L. A new signaling paradigm to control the prenylation and trafficking of small GTPases. Cell Cycle 12, 2933–2934 (2013).

    CAS  Article  Google Scholar 

  4. 4.

    Chopra, S. S. et al. Inherited CHST11/MIR3922 deletion is associated with a novel recessive syndrome presenting with skeletal malformation and malignant lymphoproliferative disease. Mol. Genet. Genom. Med. 3, 413–423 (2015).

    CAS  Article  Google Scholar 

  5. 5.

    Berghmans, S. et al. tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors. Proc. Natl Acad. Sci. USA 102, 407–412 (2005).

    CAS  Article  Google Scholar 

  6. 6.

    Spitsbergen, J. M. et al. Neoplasia in zebrafish (Danio rerio) treated with 7,12-dimethylbenz[a]anthracene by two exposure routes at different developmental stages. Toxicol. Pathol. 28, 705–715 (2000).

    CAS  Article  Google Scholar 

  7. 7.

    Li, Q. et al. Evaluation of the novel gene Rabl3 in the regulation of proliferation and motility in human cancer cells. Oncol. Rep. 24, 433–440 (2010).

    CAS  PubMed  Google Scholar 

  8. 8.

    Sowa, M. E., Bennett, E. J., Gygi, S. P. & Harper, J. W. Defining the human deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009).

    CAS  Article  Google Scholar 

  9. 9.

    Berg, T. J. et al. Splice variants of SmgGDS control small GTPase prenylation and membrane localization. J. Biol. Chem. 285, 35255–35266 (2010).

    CAS  Article  Google Scholar 

  10. 10.

    Schuld, N. J. et al. SmgGDS-558 regulates the cell cycle in pancreatic, non-small cell lung, and breast cancers. Cell Cycle 13, 941–952 (2014).

    CAS  Article  Google Scholar 

  11. 11.

    Ntantie, E. et al. An adenosine-mediated signaling pathway suppresses prenylation of the GTPase Rap1B and promotes cell scattering. Sci. Signal. 6, ra39 (2013).

    Article  Google Scholar 

  12. 12.

    Berndt, N., Hamilton, A. D. & Sebti, S. M. Targeting protein prenylation for cancer therapy. Nat. Rev. Cancer 11, 775–791 (2011).

    CAS  Article  Google Scholar 

  13. 13.

    Jones, S. et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 321, 1801–1806 (2008).

    CAS  Article  Google Scholar 

  14. 14.

    Waddell, N. et al. Whole genomes redefine the mutational landscape of pancreatic cancer. Nature 518, 495–501 (2015).

    CAS  Article  Google Scholar 

  15. 15.

    Drosten, M. et al. Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival. EMBO J. 29, 1091–1104 (2010).

    CAS  Article  Google Scholar 

  16. 16.

    Rauen, K. A. The RASopathies. Annu. Rev. Genom. Hum. Genet. 14, 355–369 (2013).

    CAS  Article  Google Scholar 

  17. 17.

    Wang, W. et al. Mice lacking Nf1 in osteochondroprogenitor cells display skeletal dysplasia similar to patients with neurofibromatosis type I. Hum. Mol. Genet. 20, 3910–3924 (2011).

    CAS  Article  Google Scholar 

  18. 18.

    Hernandez-Porras, I. et al. K-RasV14I recapitulates Noonan syndrome in mice. Proc. Natl Acad. Sci. USA 111, 16395–16400 (2014).

    CAS  Article  Google Scholar 

  19. 19.

    Roberts, N. J. et al. Whole genome sequencing defines the genetic heterogeneity of familial pancreatic cancer. Cancer Discov. 6, 166–175 (2016).

    CAS  Article  Google Scholar 

  20. 20.

    Wheeler, D. B., Zoncu, R., Root, D. E., Sabatini, D. M. & Sawyers, C. L. Identification of an oncogenic RAB. Protein Sci. 350, 211–217 (2015).

    CAS  Google Scholar 

  21. 21.

    Jindal, G. A. et al. In vivo severity ranking of Ras pathway mutations associated with developmental disorders. Proc. Natl Acad. Sci. USA 114, 510–515 (2017).

    CAS  Article  Google Scholar 

  22. 22.

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    CAS  Article  Google Scholar 

  23. 23.

    DePristo, M. A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011).

    CAS  Article  Google Scholar 

  24. 24.

    Cox, A. G. et al. Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth. Nat. Cell Biol. 18, 886–896 (2016).

    CAS  Article  Google Scholar 

  25. 25.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  Google Scholar 

  26. 26.

    Behrends, C., Sowa, M. E., Gygi, S. P. & Harper, J. W. Network organization of the human autophagy system. Nature 466, 68–76 (2010).

    CAS  Article  Google Scholar 

  27. 27.

    Huttlin, E. L. et al. The BioPlex network: a systematic exploration of the human interactome. Cell 162, 425–440 (2015).

    CAS  Article  Google Scholar 

  28. 28.

    Rauniyar, N. Parallel reaction monitoring: a targeted experiment performed using high resolution and high mass accuracy mass spectrometry. Int. J. Mol. Sci. 16, 28566–28581 (2015).

    CAS  Article  Google Scholar 

  29. 29.

    MacLean, B. et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26, 966–968 (2010).

    CAS  Article  Google Scholar 

  30. 30.

    Krieger, E. et al. Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: four approaches that performed well in CASP8. Proteins 77, 114–122 (2009).

    CAS  Article  Google Scholar 

  31. 31.

    Xu, D. & Zhang, Y. Ab initio protein structure assembly using continuous structure fragments and optimized knowledge-based force field. Proteins 80, 1715–1735 (2012).

    CAS  Article  Google Scholar 

  32. 32.

    Pierce, B. G., Hourai, Y. & Weng, Z. Accelerating protein docking in ZDOCK using an advanced 3D convolution library. PLoS ONE 6, e24657 (2011).

    CAS  Article  Google Scholar 

  33. 33.

    Ouyang, H. et al. Response of immortalized murine cementoblasts/periodontal ligament cells to parathyroid hormone and parathyroid hormone-related protein in vitro. Arch. Oral. Biol. 45, 293–303 (2000).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the NIH grant nos. K08 DK105326 (to S.N.); R01 DK090311, R01 DK095721 and R24 OD017870 (to W.G.); R01 GM095567, R01 CA157490, R01 CA188048, P01 CA117969 and R35 CA232124 (to A.C.K.); R01 CA188871 (to C.W.) and R01 GM040602 (to C.A.F.); as well as grants from the National Pancreas Foundation (to S.N.), the Harvard Digestive Diseases Center (grant no. P30 DK034854 to S.N. and W.G.), the Ken and Louise Goldberg Award (to S.N.), an ACS Research Scholar Grant (RSG-13-298-01-TBG to A.C.K.), the Lustgarten Foundation and SU2C (to A.C.K) and the Anna Fuller Fund and the Claudia Adams Barr Program for Innovative Cancer Research (to W.G.). S.N. is a recipient of the Burroughs Wellcome Fund Career Award for Medical Scientists. W.G. is a Pew Scholar in the Biomedical Sciences.

Author information

Affiliations

Authors

Contributions

S.N. and W.G. conceived and designed the overall project. S.S. and C.I.U. assisted with selecting the family, gathering the clinical histories and collecting DNA samples under human subject IRB-approved protocols. S.N., W.G. and I.L. designed the WGS analysis. I.L. performed the WGS analysis and candidate variant filtering. S.N., J.W., A.J.K., J.E.H., A.G.C. and J.H. designed and generated the zebrafish rabl3 mutant lines and performed the cancer studies. J.R.H. and S.N. performed zebrafish histology preparation and analysis. J.D.M. performed and analyzed the AP–MS experiments and CompPASS suite protein interactomics. S.N., W.G. and C.W. conceived and designed the in vitro immunoprecipitation, prenylation assays and HEK293T cell proliferation assays, and P.G., A.B., E.L. and B.U. performed these experiments. S.N. and O.M. designed and performed RASless MEF experiments. J.W.P. performed protein structural modeling. B.C.J. and C.A.F. designed and performed purification of recombinant protein. J.A.P., S.G. and J.D.M. assisted with mass spectrometry analysis. Y.H. assisted with RNA-seq data analysis. M.B.G. performed the zebrafish µCT and bone histomorphometric analysis. O.M., X.W. and J.D.M. provided assistance with tissue culture experiments. C.A.C. and J.A.R. provided analysis of clinical exome sequencing data. C.A.C. and I.L. provided analysis of variants in the Exome Aggregation Consortium. J.W.H., G.G., S.R.S., K.C. and A.C.K. provided overall input. S.N. and W.G. wrote the manuscript. All authors reviewed and edited the manuscript.

Corresponding author

Correspondence to Wolfram Goessling.

Ethics declarations

Competing interests

A.C.K. has financial interests in Vescor Therapeutics, LLC. A.C.K. is an inventor on patents pertaining to Kras-regulated metabolic pathways, redox control pathways in pancreatic cancer, targeting GOT1 as a therapeutic approach and the autophagic control of iron metabolism. A.C.K. is on the SAB of Cornerstone/Rafael Pharmaceuticals. G.G. receives research funds from IBM and Pharmacyclics. W.G. receives patent royalties from FATE Therapeutics and is on the SAB of Camp4 Therapeutics.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–19 and Supplementary Tables 3 and 5

Reporting Summary

Supplementary Table 1

Supplementary Table 1

Supplementary Table 2

Supplementary Table 2

Supplementary Table 4

Supplementary Table 4

Supplementary Video 1

Supplementary Video 1

Supplementary Video 2

Supplementary Video 2

Supplementary Video 3

Supplementary Video 3

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nissim, S., Leshchiner, I., Mancias, J.D. et al. Mutations in RABL3 alter KRAS prenylation and are associated with hereditary pancreatic cancer. Nat Genet 51, 1308–1314 (2019). https://doi.org/10.1038/s41588-019-0475-y

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing