Publications by authors named "Marielle E Yohe"

31 Publications

Immuno-transcriptomic profiling of extracranial pediatric solid malignancies.

Cell Rep 2021 Nov;37(8):110047

University of Toronto Musculoskeletal Oncology Unit, Sinai Health System; Department of Surgery, University of Toronto, Toronto, ON, Canada.

We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extracranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2021.110047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8642810PMC
November 2021

Vertical Inhibition of the RAF-MEK-ERK Cascade Induces Myogenic Differentiation, Apoptosis and Tumor Regression in H/NRAS Q61X-mutant Rhabdomyosarcoma.

Mol Cancer Ther 2021 Nov 4. Epub 2021 Nov 4.

Greehey Children's Cancer Research Institute, UTHSCSA

Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of H/NRASQ61X-mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X-dependency in RMS cells, however there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF-MEK-ERK cascade by co-suppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF-MEK-ERK cascade, and particularly co-targeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X-mutant RMS tumor growth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-21-0194DOI Listing
November 2021

Genomic Classification and Clinical Outcome in Rhabdomyosarcoma: A Report From an International Consortium.

J Clin Oncol 2021 09 24;39(26):2859-2871. Epub 2021 Jun 24.

Department of Pediatrics, Seattle Children's Hospital, Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA.

Purpose: Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. Despite aggressive therapy, the 5-year survival rate for patients with metastatic or recurrent disease remains poor, and beyond fusion status, no genomic markers are available for risk stratification. We present an international consortium study designed to determine the incidence of driver mutations and their association with clinical outcome.

Patients And Methods: Tumor samples collected from patients enrolled on Children's Oncology Group trials (1998-2017) and UK patients enrolled on malignant mesenchymal tumor and RMS2005 (1995-2016) trials were subjected to custom-capture sequencing. Mutations, indels, gene deletions, and amplifications were identified, and survival analysis was performed.

Results: DNA from 641 patients was suitable for analyses. A median of one mutation was found per tumor. In fusion-negative cases, mutation of any RAS pathway member was found in > 50% of cases, and 21% had no putative driver mutation identified. (15%), (15%), and (13%) mutations were found at a higher incidence than previously reported and mutations were associated with worse outcomes in both fusion-negative and fusion-positive cases. Interestingly, mutations in isoforms predominated in infants < 1 year (64% of cases). Mutation of was associated with histologic patterns beyond those previously described, older age, head and neck primary site, and a dismal survival. Finally, we provide a searchable companion database (ClinOmics), containing all genomic variants, and clinical annotation including survival data.

Conclusion: This is the largest genomic characterization of clinically annotated rhabdomyosarcoma tumors to date and provides prognostic genetic features that refine risk stratification and will be incorporated into prospective trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1200/JCO.20.03060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8425837PMC
September 2021

Interaction between SNAI2 and MYOD enhances oncogenesis and suppresses differentiation in Fusion Negative Rhabdomyosarcoma.

Nat Commun 2021 01 8;12(1):192. Epub 2021 Jan 8.

Greehey Children's Cancer Research Institute, Department of Molecular Medicine, University of Texas Health Sciences Center, San Antonio, Texas, USA.

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-20386-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794422PMC
January 2021

Rigosertib Induces Mitotic Arrest and Apoptosis in RAS-Mutated Rhabdomyosarcoma and Neuroblastoma.

Mol Cancer Ther 2021 02 6;20(2):307-319. Epub 2020 Nov 6.

National Cancer Institute, Bethesda, Maryland.

Relapsed pediatric rhabdomyosarcomas (RMS) and neuroblastomas (NBs) have a poor prognosis despite multimodality therapy. In addition, the current standard of care for these cancers includes vinca alkaloids that have severe toxicity profiles, further underscoring the need for novel therapies for these malignancies. Here, we show that the small-molecule rigosertib inhibits the growth of RMS and NB cell lines by arresting cells in mitosis, which leads to cell death. Our data indicate that rigosertib, like the vinca alkaloids, exerts its effects mainly by interfering with mitotic spindle assembly. Although rigosertib has the ability to inhibit oncogenic RAS signaling, we provide evidence that rigosertib does not induce cell death through inhibition of the RAS pathway in RAS-mutated RMS and NB cells. However, the combination of rigosertib and the MEK inhibitor trametinib, which has efficacy in RAS-mutated tumors, synergistically inhibits the growth of an RMS cell line, suggesting a new avenue for combination therapy. Importantly, rigosertib treatment delays tumor growth and prolongs survival in a xenograft model of RMS. In conclusion, rigosertib, through its impact on the mitotic spindle, represents a potential therapeutic for RMS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-20-0525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867632PMC
February 2021

Membrane surface recognition by the ASAP1 PH domain and consequences for interactions with the small GTPase Arf1.

Sci Adv 2020 Sep 30;6(40). Epub 2020 Sep 30.

Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA.

Adenosine diphosphate-ribosylation factor (Arf) guanosine triphosphatase-activating proteins (GAPs) are enzymes that need to bind to membranes to catalyze the hydrolysis of guanosine triphosphate (GTP) bound to the small GTP-binding protein Arf. Binding of the pleckstrin homology (PH) domain of the ArfGAP With SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) to membranes containing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] is key for maximum GTP hydrolysis but not fully understood. By combining nuclear magnetic resonance, neutron reflectometry, and molecular dynamics simulation, we show that binding of multiple PI(4,5)P molecules to the ASAP1 PH domain (i) triggers a functionally relevant allosteric conformational switch and (ii) maintains the PH domain in a well-defined orientation, allowing critical contacts with an Arf1 mimic to occur. Our model provides a framework to understand how binding of the ASAP1 PH domain to PI(4,5)P at the membrane may play a role in the regulation of ASAP1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.abd1882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527224PMC
September 2020

CASZ1 induces skeletal muscle and rhabdomyosarcoma differentiation through a feed-forward loop with MYOD and MYOG.

Nat Commun 2020 02 14;11(1):911. Epub 2020 Feb 14.

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.

Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer that expresses myogenic master regulatory factor MYOD but fails to differentiate. Here, we show that the zinc finger transcription factor CASZ1 up-regulates MYOD signature genes and induces skeletal muscle differentiation in normal myoblasts and ERMS. The oncogenic activation of the RAS-MEK pathway suppresses CASZ1 expression in ERMS. ChIP-seq, ATAC-seq and RNA-seq experiments reveal that CASZ1 directly up-regulates skeletal muscle genes and represses non-muscle genes through affecting regional epigenetic modifications, chromatin accessibility and super-enhancer establishment. Next generation sequencing of primary RMS tumors identified a single nucleotide variant in the CASZ1 coding region that potentially contributes to ERMS tumorigenesis. Taken together, loss of CASZ1 activity, due to RAS-MEK signaling or genetic alteration, impairs ERMS differentiation, contributing to RMS tumorigenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-14684-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021771PMC
February 2020

Advancing RAS/RASopathy therapies: An NCI-sponsored intramural and extramural collaboration for the study of RASopathies.

Am J Med Genet A 2020 04 8;182(4):866-876. Epub 2020 Jan 8.

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland.

RASopathies caused by germline pathogenic variants in genes that encode RAS pathway proteins. These disorders include neurofibromatosis type 1 (NF1), Noonan syndrome (NS), cardiofaciocutaneous syndrome (CFC), and Costello syndrome (CS), and others. RASopathies are characterized by heterogenous manifestations, including congenital heart disease, failure to thrive, and increased risk of cancers. Previous work led by the NCI Pediatric Oncology Branch has altered the natural course of one of the key manifestations of the RASopathy NF1. Through the conduct of a longitudinal cohort study and early phase clinical trials, the MEK inhibitor selumetinib was identified as the first active therapy for the NF1-related peripheral nerve sheath tumors called plexiform neurofibromas (PNs). As a result, selumetinib was granted breakthrough therapy designation by the FDA for the treatment of PN. Other RASopathy manifestations may also benefit from RAS targeted therapies. The overall goal of Advancing RAS/RASopathy Therapies (ART), a new NCI initiative, is to develop effective therapies and prevention strategies for the clinical manifestations of the non-NF1 RASopathies and for tumors characterized by somatic RAS mutations. This report reflects discussions from a February 2019 initiation meeting for this project, which had broad international collaboration from basic and clinical researchers and patient advocates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ajmg.a.61485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456498PMC
April 2020

Targeting RAS in pediatric cancer: is it becoming a reality?

Curr Opin Pediatr 2020 02;32(1):48-56

Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.

Purpose Of Review: The current review aims to highlight the frequency of RAS mutations in pediatric leukemias and solid tumors and to propose strategies for targeting oncogenic RAS in pediatric cancers.

Recent Findings: The three RAS genes (HRAS, NRAS, and KRAS) comprise the most frequently mutated oncogene family in human cancer. RAS mutations are commonly observed in three of the leading causes of cancer death in the United States, namely lung cancer, pancreatic cancer, and colorectal cancer. The association of RAS mutations with these aggressive malignancies inspired the creation of the National Cancer Institute RAS initiative and spurred intense efforts to develop strategies to inhibit oncogenic RAS, with much recent success. RAS mutations are frequently observed in pediatric cancers; however, recent advances in anti-RAS drug development have yet to translate into pediatric clinical trials.

Summary: We find that RAS is mutated in common and rare pediatric malignancies and that oncogenic RAS confers a functional dependency in these cancers. Many strategies for targeting RAS are being pursued for malignancies that primarily affect adults and there is a clear need for inclusion of pediatric patients in clinical trials of these agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/MOP.0000000000000856DOI Listing
February 2020

Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4,5-bisphosphate.

J Biol Chem 2019 11 6;294(46):17354-17370. Epub 2019 Oct 6.

Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892

Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP). The mechanistic basis of PIP-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP controls binding of the N-terminal extension of ARF1 to ASAP1's PH domain and thereby regulates its GAP activity. Using [Δ17]ARF1, lacking the N terminus, we found that PIP has little effect on ASAP1's activity. A soluble PIP analog, dioctanoyl-PIP (diC8PIP), stimulated GAP activity on an N terminus-containing variant, [L8K]ARF1, but only marginally affected activity on [Δ17]ARF1. A peptide comprising residues 2-17 of ARF1 ([2-17]ARF1) inhibited GAP activity, and PIP-dependently bound to a protein containing the PH domain and a 17-amino acid-long interdomain linker immediately N-terminal to the first β-strand of the PH domain. Point mutations in either the linker or the C-terminal α-helix of the PH domain decreased [2-17]ARF1 binding and GAP activity. Mutations that reduced ARF1 N-terminal binding to the PH domain also reduced the effect of ASAP1 on cellular actin remodeling. Mutations in the ARF N terminus that reduced binding also reduced GAP activity. We conclude that PIP regulates binding of ASAP1's PH domain to the ARF1 N terminus, which may partially regulate GAP activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA119.009269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873180PMC
November 2019

Targeting Glycolysis through Inhibition of Lactate Dehydrogenase Impairs Tumor Growth in Preclinical Models of Ewing Sarcoma.

Cancer Res 2019 10 20;79(19):5060-5073. Epub 2019 Aug 20.

Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both and by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis. SIGNIFICANCE: LDHA is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-19-0217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774872PMC
October 2019

Chemical genomics reveals histone deacetylases are required for core regulatory transcription.

Nat Commun 2019 07 8;10(1):3004. Epub 2019 Jul 8.

Genetics Branch, NCI, NIH, Bethesda, MD, 20892, USA.

Identity determining transcription factors (TFs), or core regulatory (CR) TFs, are governed by cell-type specific super enhancers (SEs). Drugs to selectively inhibit CR circuitry are of high interest for cancer treatment. In alveolar rhabdomyosarcoma, PAX3-FOXO1 activates SEs to induce the expression of other CR TFs, providing a model system for studying cancer cell addiction to CR transcription. Using chemical genetics, the systematic screening of chemical matter for a biological outcome, here we report on a screen for epigenetic chemical probes able to distinguish between SE-driven transcription and constitutive transcription. We find that chemical probes along the acetylation-axis, and not the methylation-axis, selectively disrupt CR transcription. Additionally, we find that histone deacetylases (HDACs) are essential for CR TF transcription. We further dissect the contribution of HDAC isoforms using selective inhibitors, including the newly developed selective HDAC3 inhibitor LW3. We show HDAC1/2/3 are the co-essential isoforms that when co-inhibited halt CR transcription, making CR TF sites hyper-accessible and disrupting chromatin looping.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-11046-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614369PMC
July 2019

Insights into pediatric rhabdomyosarcoma research: Challenges and goals.

Pediatr Blood Cancer 2019 10 21;66(10):e27869. Epub 2019 Jun 21.

Fred Hutchinson Cancer Research Center, Seattle, Washington.

Overall survival rates for pediatric patients with high-risk or relapsed rhabdomyosarcoma (RMS) have not improved significantly since the 1980s. Recent studies have identified a number of targetable vulnerabilities in RMS, but these discoveries have infrequently translated into clinical trials. We propose streamlining the process by which agents are selected for clinical evaluation in RMS. We believe that strong consideration should be given to the development of combination therapies that add biologically targeted agents to conventional cytotoxic drugs. One example of this type of combination is the addition of the WEE1 inhibitor AZD1775 to the conventional cytotoxic chemotherapeutics, vincristine and irinotecan.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pbc.27869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707829PMC
October 2019

ARF GTPases and their GEFs and GAPs: concepts and challenges.

Mol Biol Cell 2019 05;30(11):1249-1271

Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322-3050.

Detailed structural, biochemical, cell biological, and genetic studies of any gene/protein are required to develop models of its actions in cells. Studying a protein family in the aggregate yields additional information, as one can include analyses of their coevolution, acquisition or loss of functionalities, structural pliability, and the emergence of shared or variations in molecular mechanisms. An even richer understanding of cell biology can be achieved through evaluating functionally linked protein families. In this review, we summarize current knowledge of protein families: the ARF GTPases, the guanine nucleotide exchange factors (ARF GEFs) that activate them, and the GTPase-activating proteins (ARF GAPs) that have the ability to both propagate and terminate signaling. However, despite decades of scrutiny, our understanding of how these essential proteins function in cells remains fragmentary. We believe that the inherent complexity of ARF signaling and its regulation by GEFs and GAPs will require the concerted effort of many laboratories working together, ideally within a consortium to optimally pool information and resources. The collaborative study of these three functionally connected families (≥70 mammalian genes) will yield transformative insights into regulation of cell signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E18-12-0820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724607PMC
May 2019

MDS-associated mutations in germline GATA2 mutated patients with hematologic manifestations.

Leuk Res 2019 01 4;76:70-75. Epub 2018 Dec 4.

Laboratory of Myeloid Malignancies, Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

Germline mutation in GATA2 can lead to GATA2 deficiency characterized by a complex multi-system disorder that can present with many manifestations including variable cytopenias, bone marrow failure, myelodysplastic syndrome/acute myeloid leukemia (MDS/AML), and severe immunodeficiency. Penetrance and expressivity within families is often variable. There is a spectrum of bone marrow disease in symptomatic cytopenic patients ranging from hypocellular marrows without overt dysplasia to those with definitive MDS, AML, or chronic myelomonocytic leukemia. Relatives of probands with the same mutations may demonstrate minimal disease manifestations and normal marrows. A comprehensive clinical, hematological and genetic assessment of 25 patients with germline GATA2 mutation was performed. MDS-associated mutations were identified in symptomatic GATA2 patients both with overt MDS and in those with hypocellular/aplastic bone marrows without definitive dysplasia. Healthy relatives of probands harboring the same germline GATA2 mutations had essentially normal marrows that were overall devoid of MDS-associated mutations. The findings suggest that abnormal clonal hematopoiesis is a common event in symptomatic germline mutated GATA2 patients with MDS and also in those with hypocellular marrows without overt morphologic evidence of dysplasia, possibly indicating a pre-MDS stage warranting close monitoring for disease progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.leukres.2018.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340496PMC
January 2019

The tuberous sclerosis complex subunit TBC1D7 is stabilized by Akt phosphorylation-mediated 14-3-3 binding.

J Biol Chem 2018 10 24;293(42):16142-16159. Epub 2018 Aug 24.

the Structural Genomics Consortium and

The tuberous sclerosis complex (TSC) is a negative regulator of mTOR complex 1, a signaling node promoting cellular growth in response to various nutrients and growth factors. However, several regulators in TSC signaling still await discovery and characterization. Using pulldown and MS approaches, here we identified the TSC complex member, TBC1 domain family member 7 (TBC1D7), as a binding partner for PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1), a negative regulator of Akt kinase signaling. Most TBC domain-containing proteins function as Rab GTPase-activating proteins (RabGAPs), but the crystal structure of TBC1D7 revealed that it lacks residues critical for RabGAP activity. Sequence analysis identified a putative site for both Akt-mediated phosphorylation and 14-3-3 binding at Ser-124, and we found that Akt phosphorylates TBC1D7 at Ser-124. However, this phosphorylation had no effect on the binding of TBC1D7 to TSC1, but stabilized TBC1D7. Moreover, 14-3-3 protein both bound and stabilized TBC1D7 in a growth factor-dependent manner, and a phospho-deficient substitution, S124A, prevented this interaction. The crystal structure of 14-3-3ζ in complex with a phospho-Ser-124 TBC1D7 peptide confirmed the direct interaction between 14-3-3 and TBC1D7. The sequence immediately upstream of Ser-124 aligned with a canonical β-TrCP degron, and we found that the E3 ubiquitin ligase β-TrCP2 ubiquitinates TBC1D7 and decreases its stability. Our findings reveal that Akt activity determines the phosphorylation status of TBC1D7 at the phospho-switch Ser-124, which governs binding to either 14-3-3 or β-TrCP2, resulting in increased or decreased stability of TBC1D7, respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.003525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200923PMC
October 2018

MEK inhibition induces MYOG and remodels super-enhancers in RAS-driven rhabdomyosarcoma.

Sci Transl Med 2018 07;10(448)

Oncogenomics Section, Genetics Branch, National Cancer Institute, National Institutes of Health (NIH), Bethesda, MD 20892, USA.

The RAS isoforms are frequently mutated in many types of human cancers, including PAX3/PAX7 fusion-negative rhabdomyosarcoma. Pediatric RMS arises from skeletal muscle progenitor cells that have failed to differentiate normally. The role of mutant RAS in this differentiation blockade is incompletely understood. We demonstrate that oncogenic RAS, acting through the RAF-MEK [mitogen-activated protein kinase (MAPK) kinase]-ERK (extracellular signal-regulated kinase) MAPK effector pathway, inhibits myogenic differentiation in rhabdomyosarcoma by repressing the expression of the prodifferentiation myogenic transcription factor, MYOG. This repression is mediated by ERK2-dependent promoter-proximal stalling of RNA polymerase II at the locus. Small-molecule screening with a library of mechanistically defined inhibitors showed that RAS-driven RMS is vulnerable to MEK inhibition. MEK inhibition with trametinib leads to the loss of ERK2 at the promoter and releases the transcriptional stalling of expression. MYOG subsequently opens chromatin and establishes super-enhancers at genes required for late myogenic differentiation. Furthermore, trametinib, in combination with an inhibitor of IGF1R, potently decreases rhabdomyosarcoma cell viability and slows tumor growth in xenograft models. Therefore, this combination represents a potential therapeutic for RAS-mutated rhabdomyosarcoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aan4470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054766PMC
July 2018

PAX3-FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain Vulnerability.

Cancer Discov 2017 08 26;7(8):884-899. Epub 2017 Apr 26.

Genetics Branch, NCI, NIH, Bethesda, Maryland.

Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric transcription factor PAX3-FOXO1. The mechanisms by which PAX3-FOXO1 dysregulates chromatin are unknown. We find PAX3-FOXO1 reprograms the -regulatory landscape by inducing super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master transcription factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specific vulnerability that can be exploited for precision therapy. PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin-level functions are critical to understanding its oncogenic activity. We find that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-16-1297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7802885PMC
August 2017

Allosteric properties of PH domains in Arf regulatory proteins.

Cell Logist 2016 Apr-Jun;6(2):e1181700. Epub 2016 Apr 26.

Laboratory of Structural Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health , Bethesda, MD, USA.

Pleckstrin Homology (PH) domains bind phospholipids and proteins. They are critical regulatory elements of a number enzymes including guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) for Ras-superfamily guanine nucleotide binding proteins such as ADP-ribosylation factors (Arfs). Recent studies have indicated that many PH domains may bind more than one ligand cooperatively. Here we discuss the molecular basis of PH domain-dependent allosteric behavior of 2 ADP-ribosylation factor exchange factors, Grp1 and Brag2, cooperative binding of ligands to the PH domains of Grp1 and the Arf GTPase-activating protein, ASAP1, and the consequences for activity of the associated catalytic domains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/21592799.2016.1181700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878581PMC
April 2016

MultiDimensional ClinOmics for Precision Therapy of Children and Adolescent Young Adults with Relapsed and Refractory Cancer: A Report from the Center for Cancer Research.

Clin Cancer Res 2016 08 18;22(15):3810-20. Epub 2016 Mar 18.

Oncogenomics Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland.

Purpose: We undertook a multidimensional clinical genomics study of children and adolescent young adults with relapsed and refractory cancers to determine the feasibility of genome-guided precision therapy.

Experimental Design: Patients with non-central nervous system solid tumors underwent a combination of whole exome sequencing (WES), whole transcriptome sequencing (WTS), and high-density single-nucleotide polymorphism array analysis of the tumor, with WES of matched germline DNA. Clinically actionable alterations were identified as a reportable germline mutation, a diagnosis change, or a somatic event (including a single nucleotide variant, an indel, an amplification, a deletion, or a fusion gene), which could be targeted with drugs in existing clinical trials or with FDA-approved drugs.

Results: Fifty-nine patients in 20 diagnostic categories were enrolled from 2010 to 2014. Ages ranged from 7 months to 25 years old. Seventy-three percent of the patients had prior chemotherapy, and the tumors from these patients with relapsed or refractory cancers had a higher mutational burden than that reported in the literature. Thirty patients (51% of total) had clinically actionable mutations, of which 24 (41%) had a mutation that was currently targetable in a clinical trial setting, 4 patients (7%) had a change in diagnosis, and 7 patients (12%) had a reportable germline mutation.

Conclusions: We found a remarkably high number of clinically actionable mutations in 51% of the patients, and 12% with significant germline mutations. We demonstrated the clinical feasibility of next-generation sequencing in a diverse population of relapsed and refractory pediatric solid tumors. Clin Cancer Res; 22(15); 3810-20. ©2016 AACR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-15-2717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970946PMC
August 2016

Aurora B kinase is a potent and selective target in MYCN-driven neuroblastoma.

Oncotarget 2015 Nov;6(34):35247-62

Oncogenomics Section, Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed an siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of ten NB cell lines, those with MYCN-amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of the known target, histone H3, and upregulation of TP53 in MYCN-amplified, TP53 wild-type cells. However, both wild-type and TP53 mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.6208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742102PMC
November 2015

Molecular Basis for Cooperative Binding of Anionic Phospholipids to the PH Domain of the Arf GAP ASAP1.

Structure 2015 Nov 10;23(11):1977-88. Epub 2015 Sep 10.

Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Building 37, Room 2042, Bethesda, MD 20892, USA. Electronic address:

We have defined the molecular basis for association of the PH domain of the Arf GAP ASAP1 with phospholipid bilayers. Structures of the unliganded and dibutyryl PtdIns(4,5)P2-bound PH domain were solved. PtdIns(4,5)P2 made contact with both a canonical site (C site) and an atypical site (A site). We hypothesized cooperative binding of PtdIns(4,5)P2 to the C site and a nonspecific anionic phospholipid to the A site. PtdIns(4,5)P2 dependence of binding to large unilamellar vesicles and GAP activity was sigmoidal, consistent with cooperative sites. In contrast, PtdIns(4,5)P2 binding to the PH domain of PLC δ1 was hyperbolic. Mutation of amino acids in either the C or A site resulted in decreased PtdIns(4,5)P2-dependent binding to vesicles and decreased GAP activity. The results support the idea of cooperative phospholipid binding to the C and A sites of the PH domain of ASAP1. We propose that the mechanism underlies rapid switching between active and inactive ASAP1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2015.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788500PMC
November 2015

Pediatric Rhabdomyosarcoma.

Crit Rev Oncog 2015 ;20(3-4):227-43

Genetics Branch, Oncogenomics Section, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland; Pediatric Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland.

Rhabdomyosarcoma is the most common soft-tissue sarcoma of childhood, and despite clinical advances, subsets of these patients continue to suffer high levels of morbidity and mortality associated with their disease. Recent genetic and molecular characterization of these tumors using sophisticated genomics techniques, including next-generation sequencing experiments, has revealed multiple areas that can be exploited for new molecularly targeted therapies for this disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5486973PMC
http://dx.doi.org/10.1615/critrevoncog.2015013800DOI Listing
June 2016

Kras activation in p53-deficient myoblasts results in high-grade sarcoma formation with impaired myogenic differentiation.

Oncotarget 2015 Jun;6(16):14220-32

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada.

While genomic studies have improved our ability to classify sarcomas, the molecular mechanisms involved in the formation and progression of many sarcoma subtypes are unknown. To better understand developmental origins and genetic drivers involved in rhabdomyosarcomagenesis, we describe a novel sarcoma model system employing primary murine p53-deficient myoblasts that were isolated and lentivirally transduced with KrasG12D. Myoblast cell lines were characterized and subjected to proliferation, anchorage-independent growth and differentiation assays to assess the effects of transgenic KrasG12D expression. KrasG12D overexpression transformed p53-/- myoblasts as demonstrated by an increased anchorage-independent growth. Induction of differentiation in parental myoblasts resulted in activation of key myogenic regulators. In contrast, Kras-transduced myoblasts had impaired terminal differentiation. p53-/- myoblasts transformed by KrasG12D overexpression resulted in rapid, reproducible tumor formation following orthotopic injection into syngeneic host hindlimbs. Pathological analysis revealed high-grade sarcomas with myogenic differentiation based on the expression of muscle-specific markers, such as Myod1 and Myog. Gene expression patterns of murine sarcomas shared biological pathways with RMS gene sets as determined by gene set enrichment analysis (GSEA) and were 61% similar to human RMS as determined by metagene analysis. Thus, our novel model system is an effective means to model high-grade sarcomas along the RMS spectrum.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546462PMC
http://dx.doi.org/10.18632/oncotarget.3856DOI Listing
June 2015

Clonality and evolutionary history of rhabdomyosarcoma.

PLoS Genet 2015 Mar 13;11(3):e1005075. Epub 2015 Mar 13.

Genetics Branch, Oncogenomics Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America; Pediatric Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland, United States of America.

To infer the subclonality of rhabdomyosarcoma (RMS) and predict the temporal order of genetic events for the tumorigenic process, and to identify novel drivers, we applied a systematic method that takes into account germline and somatic alterations in 44 tumor-normal RMS pairs using deep whole-genome sequencing. Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype. We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1. In contrast, we find that PAX-fusion-positive (PFP) subtype tumors have undergone whole-genome duplication in the late stage of cancer evolutionary history and have acquired fewer mutations and subclones than PFN-RMS. Moreover we predict that the PAX3-FOXO1 fusion event occurs earlier than the whole genome duplication. Our findings provide information critical to the understanding of tumorigenesis of RMS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1005075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4358975PMC
March 2015

Role of the C-terminal SH3 domain and N-terminal tyrosine phosphorylation in regulation of Tim and related Dbl-family proteins.

Biochemistry 2008 Jul 7;47(26):6827-39. Epub 2008 Jun 7.

Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7295, USA.

Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho-family GTPases and typically possess tandem Dbl (DH) and pleckstrin homology (PH) domains that act in concert to catalyze exchange. Although the exchange potential of many Dbl-family proteins is constitutively activated by truncation, the precise mechanisms of regulation for many Dbl-family proteins are unknown. Tim and Vav are distantly related Dbl-family proteins that are similarly regulated; their Dbl homology (DH) domains interact with N-terminal helices to exclude and prevent activation of Rho GTPases. Phosphorylation, substitution, or deletion of the blocking helices relieves this autoinhibition. Here we show that two other Dbl-family proteins, Ngef and Wgef, which like Tim contain a C-terminal SH3 domain, are also activated by tyrosine phosphorylation of a blocking helix. Consequently, basal autoinhibition of DH domains by direct steric exclusion using short N-terminal helices likely represents a conserved mechanism of regulation for the large family of Dbl-related proteins. N-Terminal truncation or phosphorylation of many other Dbl-family GEFs leads to their activation; similar autoinhibition mechanisms could explain some of these events. In addition, we show that the C-terminal SH3 domain binding to a polyproline region N-terminal to the DH domain of the Tim subgroup of Dbl-family proteins provides a unique mechanism of regulated autoinhibition of exchange activity that is functionally linked to the interactions between the autoinhibitory helix and the DH domain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi702543pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655348PMC
July 2008

Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression.

Nat Struct Mol Biol 2007 Sep 19;14(9):814-23. Epub 2007 Aug 19.

Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.

Autoinhibition of the Rho guanine nucleotide exchange factor ASEF is relieved by interaction with the APC tumor suppressor. Here we show that binding of the armadillo repeats of APC to a 'core APC-binding' (CAB) motif within ASEF, or truncation of the SH3 domain of ASEF, relieves autoinhibition, allowing the specific activation of CDC42. Structural determination of autoinhibited ASEF reveals that the SH3 domain forms an extensive interface with the catalytic DH and PH domains to obstruct binding and activation of CDC42, and the CAB motif is positioned adjacent to the SH3 domain to facilitate activation by APC. In colorectal cancer cell lines, full-length, but not truncated, APC activates CDC42 in an ASEF-dependent manner to suppress anchorage-independent growth. We therefore propose a model in which ASEF acts as a tumor suppressor when activated by APC and inactivation of ASEF by mutation or APC truncation promotes tumorigenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb1290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2716141PMC
September 2007

Galphaq directly activates p63RhoGEF and Trio via a conserved extension of the Dbl homology-associated pleckstrin homology domain.

J Biol Chem 2007 Oct 2;282(40):29201-10. Epub 2007 Jul 2.

Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

The coordinated cross-talk from heterotrimeric G proteins to Rho GTPases is essential during a variety of physiological processes. Emerging data suggest that members of the Galpha(12/13) and Galpha(q/11) families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing Galpha(12/13)-mediated RhoA activation, proteins that functionally couple Galpha(q/11) to RhoA activation have remained elusive. Recently, the Dbl-family guanine nucleotide exchange factor (GEF) p63RhoGEF/GEFT has been described as a novel mediator of Galpha(q/11) signaling to RhoA based on its ability to synergize with Galpha(q/11) resulting in enhanced RhoA signaling in cells. We have used biochemical/biophysical approaches with purified protein components to better understand the mechanism by which activated Galpha(q) directly engages and stimulates p63RhoGEF. Basally, p63RhoGEF is autoinhibited by the Dbl homology (DH)-associated pleckstrin homology (PH) domain; activated Galpha(q) relieves this autoinhibition by interacting with a highly conserved C-terminal extension of the PH domain. This unique extension is conserved in the related Dbl-family members Trio and Kalirin and we show that the C-terminal Rho-specific DH-PH cassette of Trio is similarly activated by Galpha(q).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M703458200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655113PMC
October 2007

Auto-inhibition of the Dbl family protein Tim by an N-terminal helical motif.

J Biol Chem 2007 May 2;282(18):13813-23. Epub 2007 Mar 2.

Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599-7295, USA.

Dbl-related oncoproteins are guanine nucleotide exchange factors specific for Rho-family GTPases and typically possess tandem Dbl homology (DH) and pleckstrin homology domains that act in concert to catalyze exchange. Because the ability of many Dbl-family proteins to catalyze exchange is constitutively activated by truncations N-terminal to their DH domains, it has been proposed that the activity of Dbl-family proteins is regulated by auto-inhibition. However, the exact mechanisms of regulation of Dbl-family proteins remain poorly understood. Here we show that the Dbl-family protein, Tim, is auto-inhibited by a short, helical motif immediately N-terminal to its DH domain, which directly occludes the catalytic surface of the DH domain to prevent GTPase activation. Similar to the distantly related Vav isozymes, auto-inhibition of Tim is relieved by truncation, mutation, or phosphorylation of the auto-inhibitory helix. A peptide comprising the helical motif inhibits the exchange activity of Tim in vitro. Furthermore, substitutions within the most highly conserved surface of the DH domain designed to disrupt interactions with the auto-inhibitory helix also activate the exchange process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M700185200DOI Listing
May 2007

A Cdc42 mutant specifically activated by intersectin.

Biochemistry 2005 Oct;44(40):13282-90

Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.

The Rho family GTPase Cdc42 functions as a molecular switch and controls many fundamental cellular processes such as cytoskeletal regulation, cell polarity, and vesicular trafficking. Guanine nucleotide exchange factors of the Dbl family activate Cdc42 and other Rho GTPases by catalyzing the removal of bound GDP, allowing for GTP loading, and subsequent effector recognition ultimately leading to downstream signaling events. Analysis of existing structural data reveals that the Dbl exchange factor intersectin engages a strictly conserved GTPase residue of Cdc42 (tyrosine 32) in a unique mode with respect to all other visualized exchange factor-Rho GTPase interfaces. To investigate this differential binding architecture, we analyzed the role of tyrosine 32 of Cdc42 in binding, and stimulation by Dbl family exchange factors. Deletion of the hydroxyl side chain of tyrosine 32 substantially increases the affinity of Cdc42 for intersectin, yet severely cripples interaction with Dbs, a normally potent exchange factor of Cdc42. Moreover, Cdc42(Y32F) is exclusively activated by intersectin, while virtually unresponsive to other Cdc42-activating exchange factors in vitro and in vivo. Further, the structural determinants unique to intersectin, which permit selective recognition and concomitant stimulation of Cdc42(Y32F), have been defined. Cdc42 and other individual Rho GTPases receive input stimulatory signals from a multitude of Dbl exchange factors, and therefore, Cdc42(Y32F) could act as a valuable reagent for understanding the specific influence of ITSN on Cdc42-mediated signaling phenomena.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi050591bDOI Listing
October 2005
-->