Author 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.

No abstract

The chaperone protein SmgGDS promotes cell-cycle progression and tumorigenesis in human breast and nonsmall cell lung cancer. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, facilitate the activation of oncogenic members of the Ras and Rho families of small GTPases through membrane trafficking via regulation of the prenylation pathway. SmgGDS-607 interacts with newly synthesized preprenylated small GTPases, while SmgGDS-558 interacts with prenylated small GTPases. We determined that cancer cells have a high ratio of SmgGDS-607:SmgGDS-558 (607:558 ratio), and this elevated ratio is associated with reduced survival of breast cancer patients. These discoveries suggest that targeting SmgGDS splicing to lower the 607:558 ratio may be an effective strategy to inhibit the malignant phenotype generated by small GTPases. Here we report the development of a splice-switching oligonucleotide, named SSO Ex5, that lowers the 607:558 ratio by altering exon 5 inclusion in SmgGDS pre-mRNA (messenger RNA). Our results indicate that SSO Ex5 suppresses the prenylation of multiple small GTPases in the Ras, Rho, and Rab families and inhibits ERK activity, resulting in endoplasmic reticulum (ER) stress, the unfolded protein response, and ultimately apoptotic cell death in breast and lung cancer cell lines. Furthermore, intraperitoneal (i.p.) delivery of SSO Ex5 in MMTV-PyMT mice redirects SmgGDS splicing in the mammary gland and slows tumorigenesis in this aggressive model of breast cancer. Taken together, our results suggest that the high 607:558 ratio is required for optimal small GTPase prenylation, and validate this innovative approach of targeting SmgGDS splicing to diminish malignancy in breast and lung cancer.

Newly synthesized small GTPases in the Ras and Rho families are prenylated by cytosolic prenyltransferases and then escorted by chaperones to membranes, the nucleus, and other sites where the GTPases participate in a variety of signaling cascades. Understanding how prenylation and trafficking are regulated will help define new therapeutic strategies for cancer and other disorders involving abnormal signaling by these small GTPases. A growing body of evidence indicates that splice variants of SmgGDS (gene name RAP1GDS1) are major regulators of the prenylation, post-prenylation processing, and trafficking of Ras and Rho family members. SmgGDS-607 binds pre-prenylated small GTPases, while SmgGDS-558 binds prenylated small GTPases. This review discusses the history of SmgGDS research and explains our current understanding of how SmgGDS splice variants regulate the prenylation and trafficking of small GTPases. We discuss recent evidence that mutant forms of RabL3 and Rab22a control the release of small GTPases from SmgGDS, and review the inhibitory actions of DiRas1, which competitively blocks the binding of other small GTPases to SmgGDS. We conclude with a discussion of current strategies for therapeutic targeting of SmgGDS in cancer involving splice-switching oligonucleotides and peptide inhibitors.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest solid cancers with limited treatment options despite intensive research efforts. Familial predisposition to PDAC is thought to occur in ~10% of cases, but causative genes have not been identified in most of these families. Uncovering the genetic basis for PDAC susceptibility has immediate prognostic implications for families and can provide precious mechanistic clues to PDAC pathogenesis. Here, we perform whole-genome sequence analysis in a family with high incidence of PDAC and identify a germline nonsense mutation in the member of RAS oncogene family-like 3 (RABL3) gene that has never before been directly associated with hereditary cancer. The truncated mutant allele (RABL3_p.Ser36*) co-segregates with cancer occurrence. To evaluate the contribution of the RABL3 mutant allele in hereditary cancer, we generated rabl3 heterozygous mutant zebrafish and found increased susceptibility to cancer formation in two independent cancer models. Complementary unbiased approaches implicate RABL3 in RAS pathway regulation. RNA-Seq and genome-set enrichment analysis of juvenile rabl3 mutants reveals a KRAS upregulation signature. Furthermore, affinity-purification mass-spectrometry for proteins associated with RABL3 or RABL3_p.Ser36* identifies Rap1 GTPase-GDP Dissociation Stimulator 1 (RAP1GDS1, SmgGDS), a chaperone that regulates prenylation of RAS GTPases. Indeed, in vitro studies demonstrate that RABL3_p.Ser36* accelerates KRAS prenylation, and this impact is lost in the absence of H/N/KRAS proteins. Whereas heterozygous rabl3 mutant zebrafish exhibit cancer predisposition, homozygous rabl3 mutant zebrafish develop severe craniofacial, skeletal, and growth defects consistent with human RASopathies, and these defects are partially rescued with the MEK inhibitor trametinib. Our findings support a gain-of-function rather than a null function typically associated with premature protein truncations. The discovered causative RABL3 germline mutation provides new diagnostic opportunities for genetic testing in other cancer families and uncovers an alternative mechanism for dysregulated RAS signaling in development and cancer. Note: This abstract was not presented at the meeting. Citation Format: Sahar Nissim, Ignaty Leshchiner, Joseph D. Mancias, Matthew B. Greenblatt, Ophélia Maertens, Christopher A. Cassa, Jill A. Rosenfeld, Andrew G. Cox, John Hedgepeth, Julia Wücherpfennig, Andrew J. Kim, Jake E. Henderson, Patrick Gonyo, Anthony Brandt, Ellen Lorimer, Bethany Unger, Jeremy W. Prokop, Jeremy W. Heidel, Xiao-Xu Wang, Chinedu I. Ukaegbu, Gad Getz, Shamil R. Sunyaev, J. Wade Harper, Karen Cichowski, Alec C. Kimmelman, Yariv Houvras, Sapna Syngal, Carol Williams, Wolfram Goessling. Mutations in RABL3 alter RAS prenylation and are associated with hereditary pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4272. doi:10.1158/1538-7445.AM2017-4272

Small GTPases in the Ras and Rho families are major participants in the signaling cascades that promote malignancy. Defining how these signaling cascades are integrated can lead to new approaches to inhibit cancer development and progression. The chaperone protein SmgGDS (gene name RAP1GDS1) is perhaps the best example of a protein partner that integrates signaling by multiple small GTPases, because SmgGDS binds many Ras and Rho family members that have a C‐terminal polybasic region (PBR), including K‐Ras4B, Rap1, RhoA, RhoC, Rac1, Rac1b, DiRas1, and DiRas2. Interactions with small GTPases contribute to the well‐documented ability of SmgGDS to promote breast, lung, and prostate cancer, but our mechanistic understanding of how these interactions specifically promote malignancy is not well‐defined. We previously reported that SmgGDS controls the prenylation and membrane trafficking of newly synthesized GTPases, which promotes oncogenic signaling by the small GTPases. These results indicate that SmgGDS is a major regulator of small GTPases. We are currently investigating the alternative (but not mutually exclusive) possibility that small GTPases are major regulators of SmgGDS. In this presentation, we will discuss our recent discoveries suggesting that the binding of different small GTPases directs SmgGDS to specific subcellular compartments, where SmgGDS participates in unique signaling cascades that promote malignancy. We recently discovered that nuclear trafficking of SmgGDS causes SmgGDS to accumulate in the nucleolus and bind the nucleolar protein upstream binding factor (UBF). Our results indicate that nuclear SmgGDS protects cells from nucleolar stress and promotes cell proliferation by regulating the DREAM complex, which is a transcription factor complex that controls expression of cell cycle proteins. To begin to define whether small GTPases can regulate these functions of SmgGDS, we tested different small GTPases for their ability to control the subcellular localization and protein interactions of SmgGDS. We found that DiRas1, which is a GTPase that acts as a tumor suppressor, diminishes binding of SmgGDS to UBF and other small GTPases, and reduces nucleolar accumulation of SmgGDS, resulting in SmgGDS localizing throughout the nucleoplasm. Interestingly, RhoA expression also diminishes binding of SmgGDS to UBF, but this effect seems to be caused by RhoA diminishing nucleocytoplasmic trafficking of SmgGDS, resulting in nuclear exclusion of SmgGDS. Expression of other GTPases, such as K‐Ras4B and Rap1A, did not detectably alter SmgGDS interaction with UBF. Our discovery that specific GTPases can direct SmgGDS to different subcellular locations suggests that the interaction of PBR‐containing small GTPases with SmgGDS not only promotes unique signaling cascades mediated by the GTPases, but also promotes unique signaling cascades mediated by SmgGDS. Thus, SmgGDS might serve as both an upstream regulator, and a downstream effector, of the multiple PBR‐containing small GTPases that bind SmgGDS. These findings provide further rationale to develop therapeutic strategies targeting SmgGDS in cancer.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

To be able to fit 6 ft. of human DNA into a cell’s nucleus, an elaborate organizational system is necessary to compact the genomic DNA first into nucleosomes, and then into a 30 nm chromatin fiber. The ability to figure out the spatial conformation between these nucleosomes is vital in understanding gene regulation of underlying DNA. Literature data support two different models of the chromatin fiber that differ in their geometry and possible impact on the accessibility of the underlying DNA sequence. The “zig‐zag” model uses a straight linker DNA region, while the “solenoid” model illustrates a bent DNA linker, greatly influencing which nucleosomes are in close space in relation to one another. However, there is debate about which of these models actually represents the true structure. High resolution in vivo nucleosome maps show that the length of DNA between two nucleosomes is not constant across a genome. Computational studies have shown that changing this linker‐length can drastically change the available compacted structures accessible to an array of nucleosomes. To determine how much the properties of the linker DNA affect chromatin structure, we are developing a quantitative crosslinking assay. In our studies, we use this method to investigate the linker‐length dependence of chromatin structure in synthetic nucleosome arrays. Initial results have shown evidence for both zig‐zag and solenoid models depending on solution conditions.

Beginning with discovery of a new genetic cause of familial pancreatic ductal adenocarcinoma (PDAC), this work uncovers a mechanism for regulating KRAS intracellular trafficking with implications for hereditary cancer, sporadic cancer, and RASopathy syndromes. PDAC is one of the deadliest solid cancers, with limited treatment options despite intensive research efforts. Familial predisposition to PDAC is thought to occur in ~10% of cases, but causative genes have not been identified in most of these families. Uncovering the genetic basis for PDAC susceptibility has immediate prognostic implications for families and can provide precious mechanistic clues to PDAC pathogenesis. Here, we perform whole-genome sequence analysis in a family with five cases of PDAC and identify a germline nonsense mutation in the member of RAS oncogene family-like 3 (RABL3) gene that has never before been directly associated with hereditary cancer. The truncated mutant allele (RABL3_p.Ser36*) co-segregates with cancer occurrence. To evaluate the contribution of the RABL3 mutant allele in hereditary cancer, we generated rabl3 heterozygous mutant zebrafish and found increased susceptibility to cancer formation in two independent cancer models. Complementary unbiased approaches implicate RABL3 in RAS pathway regulation. RNA-Seq and genome-set enrichment analysis of juvenile rabl3 mutants reveals a KRAS upregulation signature. Furthermore, affinity-purification mass spectrometry for proteins associated with RABL3 or RABL3_p.Ser36* identifies Rap1 GTPase-GDP Dissociation Stimulator 1 (RAP1GDS1, SmgGDS), a chaperone that regulates prenylation of RAS GTPases. Indeed, in vitro studies demonstrate that RABL3_p.Ser36* accelerates KRAS prenylation, and this impact is lost in the absence of H/N/KRAS proteins. Whereas heterozygous rabl3 mutant zebrafish exhibit cancer predisposition, homozygous rabl3 mutant zebrafish develop severe craniofacial, skeletal, and growth defects consistent with human RASopathies, and these defects are partially rescued with the MEK inhibitor trametinib. Finally, we identify additional germline mutations in RABL3 that impact RAS activity in vivo and have a significant burden in a cohort of patients with developmental disorders, suggesting a role in undiagnosed RASopathies. Moreover, RABL3 is upregulated in multiple human PDAC cell lines and knockdown abrogates proliferation, consistent with a broader role for RABL3 in PDAC. The discovered causative RABL3 germline mutation provides new diagnostic opportunities for genetic testing in other cancer families and uncovers an alternative mechanism for dysregulated RAS signaling in development and cancer. This abstract is also being presented as Poster A12. Citation Format: Sahar Nissim, Ignaty Leshchiner, Joseph Mancias, Matthew Greenblatt, Ophélia Maertens, Christopher Cassa, Jill Rosenfeld, Andrew Cox, John Hedgepeth, Julia Wucherpfennig, Andrew Kim, Jake Henderson, Patrick Gonyo, Anthony Brandt, Ellen Lorimer, Bethany Unger, Gad Getz, Shamil Sunyaev, Wade Harper, Karen Cichowski, Alec Kimmelman, Yariv Houvras, Sapna Syngal, Carol Williams, Wolfram Goessling. Mutations in RABL3 alter KRAS prenylation and are associated with hereditary pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr PR02.