GAP-related Domain Research Articles

Genetic diagnosis of neurofibromatosis type 1: targeted next- generation sequencing with Multiple Ligation-Dependent Probe Amplification analysis

BackgroundNeurofibromatosis type 1 (NF1) is a dominantly inherited tumor predisposition syndrome that targets the peripheral nervous system. It is caused by mutations of the NF1 gene which serve as a negative regulator of the cellular Ras/MAPK (mitogen-activated protein kinases) signaling pathway. Owing to the complexity in some parts of clinical diagnoses and the need for better understanding of its molecular relationships, a genetic characterization of this disorder will be helpful in the clinical setting.MethodsIn this study, we present a customized targeted gene panel of NF1/KRAS/BRAF/p53 and SPRED1 genes combined with Multiple Ligation-Dependent Probe Amplification analysis for the NF1 mutation screening in a cohort of patients clinically suspected as NF1.ResultsIn this study, we identified 73 NF1 mutations and two BRAF novel variants from 100 NF1 patients who were suspected as having NF1. These genetic alterations are heterogeneous and distribute in a complicated way without clustering in either cysteine–serine-rich domain or within the GAP-related domain. We also detected fifteen multi-exon deletions within the NF1 gene by MLPA Analysis.ConclusionsOur results suggested that a genetic screening using a NGS panel with high coverage of Ras–signaling components combined with Multiple Ligation-Dependent Probe Amplification analysis will enable differential diagnosis of patients with overlapping clinical features.

Exhaustive non-synonymous variants functionality prediction enables high resolution characterization of the neurofibromin architecture.

BackgroundNeurofibromatosis type I (NF1) is caused by heterozygous loss-of-function variants in the NF1 gene encoding neurofibromin which serves as a tumor suppressor that inhibits RAS signaling and regulates cell proliferation and differentiation. While, the only well-established functional domain in the NF1 protein is the GAP-related domain (GRD), most of the identified non-truncating disease-causing variants are located outside of this domain, supporting the existence of other important disease-associated domains. Identifying these domains may reveal novel functions of NF1.MethodsBy implementing inferential statistics combined with machine-learning methods, we developed a novel NF1-specific functional prediction model that focuses on nonsynonymous single nucleotide variants (SNVs). The model enables annotating all possible NF1 nonsynonymous variants, thus mapping the range of pathogenic non-truncating variants at the codon level across the NF1 gene.FindingsThe generated model demonstrates high absolute prediction value for missense and splice-site variations (area under the ROC curve of 0.96) outperforming 14 other established models.By reviewing the entire dataset of nonsynonymous variants, two novel domains (Armadillo type fold 1 and 2) were identified as being associated with pathogenicity (OR 1.86; CI 1.04 to 3.34 and OR 2.08; CI 1.08 to 4.04, respectively; P < .05). Specific exons and codons associated with increased pathogenicity were also detected along the gene inside and outside the GRD domain.InterpretationThe developed model, enabled better prediction of pathogenicity for variants in NF1 gene, as well as elucidation of novel NF1-associated domains in addition to the GRD.FundThis work was partially supported by the Kahn foundation. DGE is supported by the all Manchester NIHR Biomedical Research Centre (IS-brC-1215-20007).

Abstract B43: NF1 deficiency induces aggressive mammary carcinomas in a CRISPR rat model and correlates with poor outcome in sporadic human breast cancer

Abstract Neurofibromatosis type 1 (NF1) is a common tumor predisposition syndrome caused by germline mutations in the NF1 gene that affects1 in 3,000 births. The NF1 gene encodes for neurofibromin, a GTPase that negatively regulates RAS. Women with NF1 have a 10-fold increased risk of developing breast cancer under the age of 40 and an overall lifetime risk of 18%. NF1-related breast cancers are associated with higher grade, poor prognostic factors, and poor survival (5-year survival is 68%). Comprehensive genomic analyses have also revealed that NF1 is commonly mutated in sporadic breast cancers and is a suspected driver gene. Much of our understanding of the mechanisms underlying the functional loss of NF1 comes from genetically engineered mouse models that do not completely recapitulate the phenotypes of human NF1. To examine the effect of functional NF1 loss on tumorigenesis, we induced a deletion in the NF1 GAP-related domain (GRD) in Sprague-Dawley rats using a CRISPR-Cas9 editing strategy. Because CRISPR modification results in variability at the guide site, two classes of Nf1-deficient rats were generated: larger “in-frame” indels (i.e., 63 bp) and smaller “premature stop” indels (i.e., -11). Both types of Nf1 indels induced highly penetrant and aggressive mammary adenocarcinomas in multiple rat founder lines. Mammary tumors were observed in 100% (11/11) of G0 females in mammary glands 1-5 at the average age of 51 days. Notably, two male founders also developed mammary adenocarcinomas at 14-16 months of age. This aggressive mammary phenotype was highly penetrant and multifocal mammary tumors were observed in the subsequent F1 and F2 generations from each of the Nf1 lines. Histopathologic analysis of the mammary tumors revealed that both Nf1 in-frame deletions and premature stop indels induced a wide variety of histopathologic mammary tumor types (i.e., acinar, solid, ductular, and cystic). To understand how the distinct Nf1 indels affect disease burden and survival, we examined the overall survival of females with in-frame indels (n=35) compared to premature stop indels (n=24) in three lines (Nf1IF-57/+, Nf1IF-57/PS-8, Nf1IF-54/PS 11). Kaplan-Meier analysis revealed that animals with a premature stop codon in exon 20 died due to tumor burden significantly faster than animals with an in-frame deletion (p &amp;lt; 0.0002). These results suggest that the Nf1 in-frame deletions may maintain some functional activity (compared to premature stop indels) that delays tumorigenesis. To our knowledge, this is the first model of NF1-related breast cancer and one of the few animal models of male breast cancer. Moreover, the Nf1 CRISPR rat is a novel model in which to explore the role of NF1 and deregulated Ras signaling in breast cancer initiation and progression. To examine the frequency of NF1 alterations in sporadic human breast cancer, we analyzed the METABRIC breast cancer dataset. NF1 mutations were identified in 3.8% of patients, whereas 24.5% of cancers harbored shallow deletions in NF1. Survival analysis (accounting for ER status and age at diagnosis) revealed that patients with NF1 shallow deletions are 1.65X (p &amp;lt; 1.6e-05) more likely to die within the first 10 years after diagnosis. These results reveal that NF1 deficiency may drive a more aggressive breast cancer phenotype in approximately 25% of all sporadic breast cancers. Citation Format: Patrick Dischinger, Elizabeth Tovar, Curt Essenburg, Eve Gardner, Megan Callaghan, Megan Bowman, Zachary Madaj, Ashley Turner, Anil Challa, Tristan Kempston, Bryn Eagleson, Roderick Bronson, Robert Kesterson, Matthew Steensma, Carrie Graveel. NF1 deficiency induces aggressive mammary carcinomas in a CRISPR rat model and correlates with poor outcome in sporadic human breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B43.

Computational insights of K1444N substitution in GAP-related domain of NF1 gene associated with neurofibromatosis type 1 disease: a molecular modeling and dynamics approach

The NF1 gene encodes for neurofibromin protein, which is ubiquitously expressed, but most highly in the central nervous system. Non-synonymous SNPs (nsSNPs) in the NF1 gene were found to be associated with Neurofibromatosis Type 1 disease, which is characterized by the growth of tumors along nerves in the skin, brain, and other parts of the body. In this study, we used several in silico predictions tools to analyze 16 nsSNPs in the RAS-GAP domain of neurofibromin, the K1444N (K1423N) mutation was predicted as the most pathogenic. The comparative molecular dynamic simulation (MDS; 50ns) between the wild type and the K1444N (K1423N) mutant suggested a significant change in the electrostatic potential. In addition, the RMSD, RMSF, Rg, hydrogen bonds, and PCA analysis confirmed the loss of flexibility and increase in compactness of the mutant protein. Further, SASA analysis revealed exchange between hydrophobic and hydrophilic residues from the core of the RAS-GAP domain to the surface of the mutant domain, consistent with the secondary structure analysis that showed significant alteration in the mutant protein conformation. Our data concludes that the K1444N (K1423N) mutant lead to increasing the rigidity and compactness of the protein. This study provides evidence of the benefits of the computational tools in predicting the pathogenicity of genetic mutations and suggests the application of MDS and different in silico prediction tools for variant assessment and classification in genetic clinics.

Unraveling the molecular mechanism of interactions of the Rho GTPases Cdc42 and Rac1 with the scaffolding protein IQGAP2

IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis.

Genotype-Phenotype Correlation in NF1: Evidence for a More Severe Phenotype Associated with Missense Mutations Affecting NF1 Codons 844–848

Neurofibromatosis type 1 (NF1), a common genetic disorder with a birth incidence of 1:2,000–3,000, is characterized by a highly variable clinical presentation. To date, only two clinically relevant intragenic genotype-phenotype correlations have been reported for NF1 missense mutations affecting p.Arg1809 and a single amino acid deletion p.Met922del. Both variants predispose to a distinct mild NF1 phenotype with neither externally visible cutaneous/plexiform neurofibromas nor other tumors. Here, we report 162 individuals (129 unrelated probands and 33 affected relatives) heterozygous for a constitutional missense mutation affecting one of five neighboring NF1 codons—Leu844, Cys845, Ala846, Leu847, and Gly848—located in the cysteine-serine-rich domain (CSRD). Collectively, these recurrent missense mutations affect ∼0.8% of unrelated NF1 mutation-positive probands in the University of Alabama at Birmingham (UAB) cohort. Major superficial plexiform neurofibromas and symptomatic spinal neurofibromas were more prevalent in these individuals compared with classic NF1-affected cohorts (both p < 0.0001). Nearly half of the individuals had symptomatic or asymptomatic optic pathway gliomas and/or skeletal abnormalities. Additionally, variants in this region seem to confer a high predisposition to develop malignancies compared with the general NF1-affected population (p = 0.0061). Our results demonstrate that these NF1 missense mutations, although located outside the GAP-related domain, may be an important risk factor for a severe presentation. A genotype-phenotype correlation at the NF1 region 844–848 exists and will be valuable in the management and genetic counseling of a significant number of individuals.

Ras GAP-related and C-terminal domain-dependent localization and tumorigenic activities of IQGAP1 in melanoma cells.

IQGAP1 interacts with a number of binding partners through a calponin homology domain (CHD), a WW motif, IQ repeats, a Ras GAP-related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 is known to play a role in actin cytoskeleton dynamics during membrane ruffling and lamellipodium protrusion. In addition, phosphorylation near the CT domain is thought to control IQGAP1 activity through regulation of intramolecular interaction. In a previous study, we discovered that IQGAP1 preferentially localizes to retracting areas in B16F10 mouse melanoma cells, not areas of membrane ruffling and lamellipodium protrusion. Nothing is known of the domains needed for retraction localization and very little is known of IQGAP1 function in the actin cytoskeleton of melanoma cells. Thus, we examined localization of IQGAP1 mutants to retracting areas, and characterized knock down phenotypes on tissue culture plastic and physiologic-stiffness hydrogels. Localization of IQGAP1 mutants (S1441E/S1443D, S1441A/S1443A, ΔCHD, ΔGRD or ΔCT) to retracting and protruding cell edges were measured. In retracting areas there was a decrease in S1441A/S1443A, ΔGRD and ΔCT localization, a minor decrease in ΔCHD localization, and normal localization of the S1441E/S1443D mutant. In areas of cell protrusion just behind the lamellipodium leading edge, we surprisingly observed both ΔGRD and ΔCT localization, and increased number of microtubules. IQGAP1 knock down caused loss of cell polarity on laminin-coated glass, decreased proliferation on tissue culture polystyrene, and abnormal spheroid growth on laminin-coated hydrogels. We propose that the GRD and CT domains regulate IQGAP1 localization to retracting actin networks to promote a tumorigenic role in melanoma cells.

NMR resonance assignments of the EVH1 domain of neurofibromin\u2019s recruitment factor Spred1

Neurofibromin and Sprouty-related EVH1 domain-containing protein 1 (Spred1) both act as negative regulators of the mitogen-activated protein kinase pathway and are associated with the rare diseases Neurofibromatosis type 1 and Legius syndrome, respectively. Spred1 recruits the major GTPase activating protein (GAP) neurofibromin from the cytosol to the membrane in order to inactivate the small G protein Ras. These functions are dependent on the N-terminal EVH1 domain and the C-terminal Sprouty domain of Spred1 whereas the former specifically recognizes the GAP related domain of neurofibromin and the latter is responsible for membrane targeting. Within the GAP domain, Spred1 binding depends on the GAPex portion which is dispensable for Ras inactivation. In a first step towards the characterization of the Neurofibromin Spred1 interface in solution we assigned backbone and side chain 1H, 13C, and 15N chemical shifts of the Spred1 derived EVH1 domain. Our chemical shift data analysis indicate seven consecutive β-strands followed by a C-terminal α-helix which is in agreement with the previously reported crystal structure of Spred1(EVH1). Our data provide a framework for further analysis of the function of patient-derived mutations associated with rare diseases.

Abstract 1351: Alternative splicing of neurofibromin 1 is associated with elevated MAPK activity and poor prognosis in glioma

Abstract High-grade gliomas (HGG) are invasive with poor prognosis regardless of age: diffuse intrinsic pontine gliomas (DIPG), arising in the brainstem, are almost universally fatal and the leading cause of brain-tumor death in children; while adult anaplastic astrocytoma and glioblastoma multiforme (GBM) have median survivals of 1-3 years. The mutational spectra of adult and pediatric HGG differ, with pediatric tumours containing recurrent mutations of H3F3A and HIST1H3B. However, alterations leading to RAS/MAPK/PI3K pathway activation, including PDGFRA amplification, EGFRvIII, BRAF-V600E, NF1 deletion, are frequently found, although not all tumours will have mutations in this pathway. The neurofibromin 1 (NF1) gene negatively regulates RAS signalling by stimulating RAS-GTP turnover, thereby leading to RAS-inactivation. The two major isoforms, NF1-I and NF1-II, differ only by inclusion of the 21 aa exon23a in the GAP-related domain of NF1-II. Exon23a-inclusion has been shown to render NF1 10 times less active towards RAS, leading to elevated MAPK signalling. The brain expresses predominantly NF1-I, while the major isoform elsewhere is NF1-II. Here we used RNA-Seq to identify genes alternatively spliced between DIPG and normal brain, identifying an isoform switch from NF1-I in normal brain to NF1-II; we additionally found the same isoform switch in the TCGA adult GBM and LGG cohorts. For both GBM and LGG, RAS/MAPK/PI3K wild-type tumors with elevated NF1-II conferred significantly reduced patient survival compared to RAS/MAPK/PI3K mutant tumors. NF1-exon23a inclusion is known to be repressed in cell model systems by the CELF and ELAV-like families of splice regulators. We further show that members of these gene families are downregulated in HGG. Together, our results indicate a novel mechanism by which gliomas can activate signaling downstream from RAS independent of mutations and tumor grade, which promotes tumorigenesis by regulating pathways such as proliferation and invasion. Citation Format: Robert Siddaway, Arun Ramani, Man Yu, Michael Brudno, Cynthia Hawkins. Alternative splicing of neurofibromin 1 is associated with elevated MAPK activity and poor prognosis in glioma [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 1351. doi:10.1158/1538-7445.AM2017-1351

Abnormal Neural Progenitor Cells Differentiated from Induced Pluripotent Stem Cells Partially Mimicked Development of TSC2 Neurological Abnormalities.

SummaryTuberous sclerosis complex (TSC) is a disease featuring devastating and therapeutically challenging neurological abnormalities. However, there is a lack of specific neural progenitor cell models for TSC. Here, the pathology of TSC was studied using primitive neural stem cells (pNSCs) from a patient presenting a c.1444-2A>C mutation in TSC2. We found that TSC2 pNSCs had higher proliferative activity and increased PAX6 expression compared with those of control pNSCs. Neurons differentiated from TSC2 pNSCs showed enlargement of the soma, perturbed neurite outgrowth, and abnormal connections among cells. TSC2 astrocytes had increased saturation density and higher proliferative activity. Moreover, the activity of the mTOR pathway was enhanced in pNSCs and induced in neurons and astrocytes. Thus, our results suggested that TSC2 heterozygosity caused neurological malformations in pNSCs, indicating that its heterozygosity might be sufficient for the development of neurological abnormalities in patients.

IQGAP1 Interaction with RHO Family Proteins Revisited: KINETIC AND EQUILIBRIUM EVIDENCE FOR MULTIPLE DISTINCT BINDING SITES

IQ motif-containing GTPase activating protein 1 (IQGAP1) plays a central role in the physical assembly of relevant signaling networks that are responsible for various cellular processes, including cell adhesion, polarity, and transmigration. The RHO family proteins CDC42 and RAC1 have been shown to mainly interact with the GAP-related domain (GRD) of IQGAP1. However, the role of its RASGAP C-terminal (RGCT) and C-terminal domains in the interactions with RHO proteins has remained obscure. Here, we demonstrate that IQGAP1 interactions with RHO proteins underlie a multiple-step binding mechanism: (i) a high affinity, GTP-dependent binding of RGCT to the switch regions of CDC42 or RAC1 and (ii) a very low affinity binding of GRD and a C terminus adjacent to the switch regions. These data were confirmed by phosphomimetic mutation of serine 1443 to glutamate within RGCT, which led to a significant reduction of IQGAP1 affinity for CDC42 and RAC1, clearly disclosing the critical role of RGCT for these interactions. Unlike CDC42, an extremely low affinity was determined for the RAC1-GRD interaction, suggesting that the molecular nature of IQGAP1 interaction with CDC42 partially differs from that of RAC1. Our study provides new insights into the interaction characteristics of IQGAP1 with RHO family proteins and highlights the complementary importance of kinetic and equilibrium analyses. We propose that the ability of IQGAP1 to interact with RHO proteins is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of IQGAP1-mediated cellular responses.

The Structural Basis for Cdc42-Induced Dimerization of IQGAPs

In signaling, Rho-family GTPases bind effector proteins and alter their behavior. Here we present the crystal structure of Cdc42·GTP bound to the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP2. Four molecules of Cdc42 are bound to two GRD molecules, which bind each other in a parallel dimer. Two Cdc42s bind very similarly to the Ras/RasGAP interaction, while the other two bind primarily to "extra domain" sequences from both GRDs, tying the GRDs together. Calorimetry confirms two-site binding of Cdc42·GTP for the GRDs of both IQGAP2 and IQGAP1. Mutation of important extra domain residues reduces binding to single-site and abrogates Cdc42 binding to a much larger IQGAP1 fragment. Importantly, Rac1·GTP displays only single-site binding to the GRDs, indicating that only Cdc42 promotes IQGAP dimerization. The structure identifies an unexpected role for Cdc42 in protein dimerization, thus expanding the repertoire of interactions of Ras family proteins with their targets.

Abstract 2456: Synergy between loss of NF1 and overexpression of MYCN in neuroblastoma is mediated by the GAP-related domain

Abstract Earlier reports indicated that the role of Nf1 tumor suppressor gene in limiting sympathoadrenal cell growth during embryologic development is independent of its ability to down-modulate RAS-MAPK signaling. This finding raised the question of whether neuroblastoma pathogenesis was also accelerated by loss of a similar non-canonical function of NF1. To elucidate how loss of the NF1 tumor suppressor gene contributes to the development of high-risk neuroblastoma, we relied on a transgenic zebrafish model that overexpresses MYCN and harbors loss-of-function nf1 mutations. We show here that loss of nf1 leads to aberrant activation of RAS signaling in MYCN-induced neuroblastoma, promoting both increased tumor cell survival and rapid tumor cell proliferation. We demonstrate further that the GTPase-activating protein (GAP) activity of the (GAP)-related domain (GRD) is sufficient to suppress accelerated initiation of neuroblastoma in nf1-deficient zebrafish, even though this transgene is unable to restrict abnormal sympathoadrenal cell growth during embryologic development. Hence NF1 exhibits different activities in vivo in the normal development and tumorigenesis of the peripheral sympathetic nervous system. Our findings establish nf1-deficient zebrafish that overexpress MYCN as an ideal animal model system for investigating new strategies to improve treatment of very high risk neuroblastomas with aberrant RAS-MAPK activation. We are currently performing high-throughput in vivo drug analysis using these zebrafish with primary tumors. Citation Format: Shuning He, Marc R. Mansour, Mark W. Zimmerman, Dong Hyuk Ki, Hillary M. Layden, Koshi Akahane, Eric D. de Groh, Antonio R. Perez-Atayde, Shizhen Zhu, Jonathan A. Epstein, A Thomas Look. Synergy between loss of NF1 and overexpression of MYCN in neuroblastoma is mediated by the GAP-related domain. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2456.

Abstract 19: Neurofibromatosis type 1 (NF1) status determines sensitivity of soft tissue sarcoma and melanoma cell lines to glutaminase inhibitors

Abstract Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic syndrome caused by a mutation in or deletion of the NF1 gene. Mutations in the NF1 gene lead to the production of a nonfunctional version of neurofibromin that cannot regulate cell growth and division. As a result, tumors such as neurofibromas can form along nerves throughout the body. The NF1 phenotype is highly penetrant and occurs in 1 in 3,000 to 4,000 people worldwide. Individuals with an altered NF1 gene are at an increased risk of developing benign and/or malignant tumors. NF1 has been shown to negatively regulate Ras activity. Ras-driven cancer cells have also been known to alter glucose and glutamine metabolism. In the present study, we evaluated the role played by NF1 status in determining the sensitivity of sarcoma and melanoma cell lines to glutaminase inhibition and its effect on Ras activity. We tested a panel of soft tissue sarcoma and melanoma cell lines that were either wild-type, null or mutant for NF1. Results from our in vitro proliferation assay showed that compared to wild-type NF1 (STS26T, LS141) cell lines, NF1 mutant (MPNST) and NF1 null (ST88 and MeWo) cell lines showed greater sensitivity to inhibition of proliferation by glutaminase inhibitors CB-839 and BPTES. Western blot analysis showed induction of apoptosis and down regulation of mTORC1 targets such as phospho-S6K and phospho-S6 by glutaminase inhibitors only in NF1 null and NF1 mutant but not wild-type NF1 cell lines. Gene silencing experiments showed that siRNA mediated knockdown of NF1 sensitizes LS141and STS26T cell lines to glutaminase inhibition. Conversely, overexpression of wild-type NF1 GRD (GAP related domain) in MeWo cell line resulted in decreased sensitivity to glutaminase inhibition when tested in a cell proliferation assay, thus, confirming the role played by NF1. Previous reports have shown that mutation or deletion of NF1 results in activation of Ras. In order to test the effect of glutaminase inhibition on Ras activity, we carried out Ras-GTP pull down assay following the treatment with glutaminase inhibitors in NF1 null and wild-type NF1 cell lines. Our results showed that glutaminase inhibition leads to down regulation of activated RAS in NF1 null but not wild-type NF1 cells. SiRNA mediated knockdown of NF1 followed by glutaminase inhibition in wild-type NF1 cell line (LS141) resulted in decreased Ras activity, further confirming our hypothesis. Results from patient derived MPNST tumor xenograft model showed a significant suppression of tumor volume when tumors were treated with glutaminase inhibitor compared to vehicle control. Taken together, our data strongly indicates that NF1 status determines the sensitivity of sarcoma and melanoma cell lines to glutaminase inhibition. Further research is warranted to explore glutaminase inhibition as potential therapy for patients with NF1 loss and/or mutation. Citation Format: Tahir N. Sheikh, Parag P. Patwardhan, Gary K. Schwartz. Neurofibromatosis type 1 (NF1) status determines sensitivity of soft tissue sarcoma and melanoma cell lines to glutaminase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 19.

The neurofibromin recruitment factor Spred1 binds to the GAP related domain without affecting Ras inactivation

Neurofibromatosis type 1 (NF1) and Legius syndrome are related diseases with partially overlapping symptoms caused by alterations of the tumor suppressor genes NF1 (encoding the protein neurofibromin) and SPRED1 (encoding sprouty-related, EVH1 domain-containing protein 1, Spred1), respectively. Both proteins are negative regulators of Ras/MAPK signaling with neurofibromin functioning as a Ras-specific GTPase activating protein (GAP) and Spred1 acting on hitherto undefined components of the pathway. Importantly, neurofibromin has been identified as a key protein in the development of cancer, as it is genetically altered in a large number of sporadic human malignancies unrelated to NF1. Spred1 has previously been demonstrated to interact with neurofibromin via its N-terminal Ena/VASP Homology 1 (EVH1) domain and to mediate membrane translocation of its target dependent on its C-terminal Sprouty domain. However, the region of neurofibromin required for the interaction with Spred1 has remained unclear. Here we show that the EVH1 domain of Spred1 binds to the noncatalytic (GAPex) portion of the GAP-related domain (GRD) of neurofibromin. Binding is compatible with simultaneous binding of Ras and does not interfere with GAP activity. Our study points to a potential targeting function of the GAPex subdomain of neurofibromin that is present in all known canonical RasGAPs.

Synergy between loss of NF1 and overexpression of MYCN in neuroblastoma is mediated by the GAP-related domain.

Earlier reports showed that hyperplasia of sympathoadrenal cell precursors during embryogenesis in Nf1-deficient mice is independent of Nf1's role in down-modulating RAS-MAPK signaling. We demonstrate in zebrafish that nf1 loss leads to aberrant activation of RAS signaling in MYCN-induced neuroblastomas that arise in these precursors, and that the GTPase-activating protein (GAP)-related domain (GRD) is sufficient to suppress the acceleration of neuroblastoma in nf1-deficient fish, but not the hypertrophy of sympathoadrenal cells in nf1 mutant embryos. Thus, even though neuroblastoma is a classical "developmental tumor", NF1 relies on a very different mechanism to suppress malignant transformation than it does to modulate normal neural crest cell growth. We also show marked synergy in tumor cell killing between MEK inhibitors (trametinib) and retinoids (isotretinoin) in primary nf1a-/- zebrafish neuroblastomas. Thus, our model system has considerable translational potential for investigating new strategies to improve the treatment of very high-risk neuroblastomas with aberrant RAS-MAPK activation.

Interaction between a Domain of the Negative Regulator of the Ras-ERK Pathway, SPRED1 Protein, and the GTPase-activating Protein-related Domain of Neurofibromin Is Implicated in Legius Syndrome and Neurofibromatosis Type 1

Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple café-au-lait macules, axillary freckling, learning disabilities, and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the Ras-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids and the C-terminal 20 amino acids of the GTPase-activating protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing α-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120(GAP). Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome.

Translationally Controlled Tumor Protein Is a Novel Biological Target for Neurofibromatosis Type 1-associated Tumors

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that predisposes individuals to develop benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). Due to the lack of information on the molecular mechanism of NF1-associated tumor pathogenesis or biomarkers/therapeutic targets, an effective treatment for NF1 tumors has not been established. In this study, the novel NF1-associated protein, translationally controlled tumor protein (TCTP), was identified by integrated proteomics and found to be up-regulated via activated MAPK/PI3K-AKT signaling in response to growth factors in NF1-deficient Schwann cells. Immunohistochemical analysis of NF1-associated tumors revealed that the TCTP expression level correlated with tumorigenicity. In NF1-deficient MPNST cells, TCTP protein but not mRNA was down-regulated by NF1 GTPase-activating protein-related domain or MAPK/PI3K inhibitors, and this correlated with suppression of mammalian target of rapamycin (mTOR) signaling. mTOR inhibition by rapamycin also down-regulated TCTP protein expression, whereas knockdown or overexpression of TCTP suppressed or activated mTOR signaling, respectively, and affected cell viability. These results suggest that a positive feedback loop between TCTP and mTOR contributes to NF1-associated tumor formation. Last, the anti-tumor effect of artesunate, which binds to and degrades TCTP, was evaluated. Artesunate significantly suppressed the viability of MPNST cells but not normal Schwann cells, and the TCTP level inversely correlated with artesunate sensitivity. Moreover, combinational use of artesunate and rapamycin enhanced the cytotoxic effect on MPNST cells. These findings suggest that TCTP is functionally implicated in the progression of NF1-associated tumors and could serve as a biological target for their therapy.

Characterization of Recombinant ELMOD (Cell Engulfment and Motility Domain) Proteins as GTPase-activating Proteins (GAPs) for ARF Family GTPases

The ARF family of regulatory GTPases, within the RAS superfamily, is composed of ~30 members in mammals, including up to six ARF and at least 18 ARF-like (ARL) proteins. They exhibit significant structural and biochemical conservation and regulate a variety of essential cellular processes, including membrane traffic, cell division, and energy metabolism; each with links to human diseases. We previously identified members of the ELMOD family as GTPase-activating proteins (GAPs) for ARL2 that displayed crossover activity for ARFs as well. To further characterize the GAP activities of the three human ELMODs as GAPs we developed new preparations of each after overexpression in human embryonic kidney (HEK293T) cells. This allowed much higher specific activities and enhanced stability and solubility of the purified proteins. The specificities of ELMOD1-3 as GAPs for six different members of the ARF family were determined and found to display wide variations, which we believe will reveal differences in cellular functions of family members. The non-opioid sigma-1 receptor (S1R) was identified as a novel effector of GAP activity of ELMOD1-3 proteins as its direct binding to either ELMOD1 or ELMOD2 resulted in loss of GAP activity. These findings are critical to understand the roles of ELMOD proteins in cell signaling in general and in the inner ear specifically, and open the door to exploration of the regulation of their GAP activities via agonists or antagonists of the S1R.

IQGAP1 is a novel phosphatidylinositol 4,5 bisphosphate effector in regulation of directional cell migration

Phosphatidylinositol 4,5 bisphosphate (PIP₂) is a key lipid messenger for regulation of cell migration. PIP₂ modulates many effectors, but the specificity of PIP₂ signalling can be defined by interactions of PIP₂-generating enzymes with PIP₂ effectors. Here, we show that type Iγ phosphatidylinositol 4-phosphate 5-kinase (PIPKIγ) interacts with the cytoskeleton regulator, IQGAP1, and modulates IQGAP1 function in migration. We reveal that PIPKIγ is required for IQGAP1 recruitment to the leading edge membrane in response to integrin or growth factor receptor activation. Moreover, IQGAP1 is a PIP₂ effector that directly binds PIP₂ through a polybasic motif and PIP₂ binding activates IQGAP1, facilitating actin polymerization. IQGAP1 mutants that lack PIPKIγ or PIP₂ binding lose the ability to control directional cell migration. Collectively, these data reveal a synergy between PIPKIγ and IQGAP1 in the control of cell migration.