Publications by authors named "Steven S Rosenfeld"

56 Publications

Protein kinase C and SRC signaling define reciprocally related subgroups of glioblastoma with distinct therapeutic vulnerabilities.

Cell Rep 2021 Nov;37(8):110054

Department of Cancer Biology, Mayo Clinic, Jacksonville, FL 32224, USA. Electronic address:

We report that atypical protein kinase Cι (PKCι) is an oncogenic driver of glioblastoma (GBM). Deletion or inhibition of PKCι significantly impairs tumor growth and prolongs survival in murine GBM models. GBM cells expressing elevated PKCι signaling are sensitive to PKCι inhibitors, whereas those expressing low PKCι signaling exhibit active SRC signaling and sensitivity to SRC inhibitors. Resistance to the PKCι inhibitor auranofin is associated with activated SRC signaling and response to a SRC inhibitor, whereas resistance to a SRC inhibitor is associated with activated PKCι signaling and sensitivity to auranofin. Interestingly, PKCι- and SRC-dependent cells often co-exist in individual GBM tumors, and treatment of GBM-bearing mice with combined auranofin and SRC inhibitor prolongs survival beyond either drug alone. Thus, we identify PKCι and SRC signaling as distinct therapeutic vulnerabilities that are directly translatable into an improved treatment for GBM.
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http://dx.doi.org/10.1016/j.celrep.2021.110054DOI Listing
November 2021

Flexible microtubule anchoring modulates the bi-directional motility of the kinesin-5 Cin8.

Cell Mol Life Sci 2021 Aug 17;78(16):6051-6068. Epub 2021 Jul 17.

Department of Chemistry, Ben-Gurion University of the Negev, 84105, Beer-Sheva, Israel.

Two modes of motility have been reported for bi-directional kinesin-5 motors: (a) context-dependent directionality reversal, a mode in which motors undergo persistent minus-end directed motility at the single-molecule level and switch to plus-end directed motility in different assays or under different conditions, such as during MT gliding or antiparallel sliding or as a function of motor clustering; and (b) bi-directional motility, defined as movement in two directions in the same assay, without persistent unidirectional motility. Here, we examine how modulation of motor-microtubule (MT) interactions affects these two modes of motility for the bi-directional kinesin-5, Cin8. We report that the large insert in loop 8 (L8) within the motor domain of Cin8 increases the MT affinity of Cin8 in vivo and in vitro and is required for Cin8 intracellular functions. We consistently found that recombinant purified L8 directly binds MTs and L8 induces single Cin8 motors to behave according to context-dependent directionality reversal and bi-directional motility modes at intermediate ionic strength and according to a bi-directional motility mode in an MT surface-gliding assay under low motor density conditions. We propose that the largely unstructured L8 facilitates flexible anchoring of Cin8 to the MTs. This flexible anchoring enables the direct observation of bi-directional motility in motility assays. Remarkably, although L8-deleted Cin8 variants exhibit a strong minus-end directed bias at the single-molecule level, they also exhibit plus-end directed motility in an MT-gliding assay. Thus, L8-induced flexible MT anchoring is required for bi-directional motility of single Cin8 molecules but is not necessary for context-dependent directionality reversal of Cin8 in an MT-gliding assay.
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http://dx.doi.org/10.1007/s00018-021-03891-xDOI Listing
August 2021

Analysis of intraoperative human brain tissue transcriptome reveals putative risk genes and altered molecular pathways in glioma-related seizures.

Epilepsy Res 2021 Jul 18;173:106618. Epub 2021 Mar 18.

Department of Neurosurgery, Mayo Clinic, Jacksonville, FL, USA. Electronic address:

Background: The pathogenesis of glioma-related seizures (GRS) is poorly understood. Here in, we aim to identify putative molecular pathways that lead to the development of GRS.

Methods: We determined brain transcriptome from intraoperative human brain tissue of patients with either GRS, glioma without seizures (non-GRS), or with idiopathic temporal lobe epilepsy (iTLE). We performed transcriptome-wide comparisons between disease groups tissue from non-epileptic controls (non-EC) to identify differentially-expressed genes (DEG). We compared DEGs to identify those that are specific or common to the groups. Through a gene ontology analysis, we identified molecular pathways enriched for genes with a Log-fold change ≥1.5 or ≤-1.5 and p-value <0.05 compared to non-EC.

Results: We identified 110 DEGs that are associated with GRS vs. non-GRS: 80 genes showed high and 30 low expression in GRS. There was significant overexpression of genes involved in cell-to-cell and glutamatergic signaling (CELF4, SLC17A7, and CAMK2A) and down-regulation of genes involved immune-trafficking (CXCL8, H19, and VEGFA). In the iTLE vs GRS analysis, there were 1098 DEGs: 786 genes were overexpressed and 312 genes were underexpressed in the GRS samples. There was significant enrichment for genes considered markers of oncogenesis (GSC, MYBL2, and TOP2A). Further, there was down-regulation of genes involved in the glutamatergic neurotransmission (vesicular glutamate transporter-2) in the GRS vs. iTLE samples.

Conclusions: We identified a number of altered processes such as cell-to-cell signaling and interaction, inflammation-related, and glutamatergic neurotransmission in the pathogenesis of GRS. Our findings offer a new landscape of targets to further study in the fields of brain tumors and seizures.
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http://dx.doi.org/10.1016/j.eplepsyres.2021.106618DOI Listing
July 2021

Myosin 10 Regulates Invasion, Mitosis, and Metabolic Signaling in Glioblastoma.

iScience 2020 Dec 13;23(12):101802. Epub 2020 Nov 13.

Department of Cancer Biology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA.

Invasion and proliferation are defining phenotypes of cancer, and in glioblastoma blocking one stimulates the other, implying that effective therapy must inhibit both, ideally through a single target that is also dispensable for normal tissue function. The molecular motor myosin 10 meets these criteria. Myosin 10 knockout mice can survive to adulthood, implying that normal cells can compensate for its loss; its deletion impairs invasion, slows proliferation, and prolongs survival in murine models of glioblastoma. Myosin 10 deletion also enhances tumor dependency on the DNA damage and the metabolic stress responses and induces synthetic lethality when combined with inhibitors of these processes. Our results thus demonstrate that targeting myosin 10 is active against glioblastoma by itself, synergizes with other clinically available therapeutics, may have acceptable side effects in normal tissues, and has potential as a heretofore unexplored therapeutic approach for this disease.
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http://dx.doi.org/10.1016/j.isci.2020.101802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702012PMC
December 2020

The mechanism of kinesin inhibition by kinesin-binding protein.

Elife 2020 11 30;9. Epub 2020 Nov 30.

Institute of Structural and Molecular Biology, Birkbeck, University of London, London, United Kingdom.

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin-binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment, and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a tetratricopeptide repeat-containing, right-handed α-solenoid that sequesters the kinesin motor domain's tubulin-binding surface, structurally distorting the motor domain and sterically blocking its MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain, and selected structure-guided mutations disrupt KBP inhibition of kinesin transport in cells. The KBP-interacting motor domain surface contains motifs exclusively conserved in KBP-interacting kinesins, suggesting a basis for kinesin selectivity.
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http://dx.doi.org/10.7554/eLife.61481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746232PMC
November 2020

Enhancing Brain Retention of a KIF11 Inhibitor Significantly Improves its Efficacy in a Mouse Model of Glioblastoma.

Sci Rep 2020 04 16;10(1):6524. Epub 2020 Apr 16.

Brain Barriers Research Center, Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, MN, USA.

Glioblastoma, the most lethal primary brain cancer, is extremely proliferative and invasive. Tumor cells at tumor/brain-interface often exist behind a functionally intact blood-brain barrier (BBB), and so are shielded from exposure to therapeutic drug concentrations. An ideal glioblastoma treatment needs to engage targets that drive proliferation as well as invasion, with brain penetrant therapies. One such target is the mitotic kinesin KIF11, which can be inhibited with ispinesib, a potent molecularly-targeted drug. Although, achieving durable brain exposures of ispinesib is critical for adequate tumor cell engagement during mitosis, when tumor cells are vulnerable, for efficacy. Our results demonstrate that the delivery of ispinesib is restricted by P-gp and Bcrp efflux at BBB. Thereby, ispinesib distribution is heterogeneous with concentrations substantially lower in invasive tumor rim (intact BBB) compared to glioblastoma core (disrupted BBB). We further find that elacridar-a P-gp and Bcrp inhibitor-improves brain accumulation of ispinesib, resulting in remarkably reduced tumor growth and extended survival in a rodent model of glioblastoma. Such observations show the benefits and feasibility of pairing a potentially ideal treatment with a compound that improves its brain accumulation, and supports use of this strategy in clinical exploration of cell cycle-targeting therapies in brain cancers.
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http://dx.doi.org/10.1038/s41598-020-63494-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162859PMC
April 2020

The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding.

Elife 2020 01 20;9. Epub 2020 Jan 20.

Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States.

Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.
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http://dx.doi.org/10.7554/eLife.51131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015671PMC
January 2020

Myosin IIA suppresses glioblastoma development in a mechanically sensitive manner.

Proc Natl Acad Sci U S A 2019 07 24;116(31):15550-15559. Epub 2019 Jun 24.

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Jacksonville, FL 32224;

The ability of glioblastoma to disperse through the brain contributes to its lethality, and blocking this behavior has been an appealing therapeutic approach. Although a number of proinvasive signaling pathways are active in glioblastoma, many are redundant, so targeting one can be overcome by activating another. However, these pathways converge on nonredundant components of the cytoskeleton, and we have shown that inhibiting one of these-the myosin II family of cytoskeletal motors-blocks glioblastoma invasion even with simultaneous activation of multiple upstream promigratory pathways. Myosin IIA and IIB are the most prevalent isoforms of myosin II in glioblastoma, and we now show that codeleting these myosins markedly impairs tumorigenesis and significantly prolongs survival in a rodent model of this disease. However, while targeting just myosin IIA also impairs tumor invasion, it surprisingly increases tumor proliferation in a manner that depends on environmental mechanics. On soft surfaces myosin IIA deletion enhances ERK1/2 activity, while on stiff surfaces it enhances the activity of NFκB, not only in glioblastoma but in triple-negative breast carcinoma and normal keratinocytes as well. We conclude myosin IIA suppresses tumorigenesis in at least two ways that are modulated by the mechanics of the tumor and its stroma. Our results also suggest that inhibiting tumor invasion can enhance tumor proliferation and that effective therapy requires targeting cellular components that drive both proliferation and invasion simultaneously.
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http://dx.doi.org/10.1073/pnas.1902847116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681735PMC
July 2019

Progress Toward Long-Term Survivors of Glioblastoma.

Mayo Clin Proc 2019 07 20;94(7):1278-1286. Epub 2019 Jun 20.

Department of Radiation Oncology, Mayo Clinic, Jacksonville, FL; Division of Biomedical Statistics and Informatics, Mayo Clinic, Jacksonville, FL. Electronic address:

Objective: To identify the frequency and characteristics of long-term survivors of glioblastoma.

Patients And Methods: Using all cases of glioblastoma with histopathological confirmation in the National Cancer Database from January 1, 2004, through December 31, 2009, clinical, institutional, and treatment-related factors were evaluated with multivariable logistic regression models so as to elucidate factors independently associated with higher than 5-year overall survival after diagnosis.

Results: A total of 48,652 patients met the inclusion criteria, with 2249 (4.6%) achieving 5-year survival. Factors associated with odds of improved 5-year overall survival in multivariable analysis were younger age, female sex, less medical comorbidities, nonwhite race, highest median income quartile, left-sided tumors and tumors outside the brainstem, and treatment with radiotherapy (P<.05 for all). The percentage of patients surviving 5 years remained relatively unchanged over the 6-year study period (P=.97).

Conclusion: Despite improvements in median and short-term overall survival shown in recent large clinical trials for glioblastoma, the percentage of patients with glioblastoma achieving 5-year overall survival remains low. This observation calls for the development of practice-redefining therapies and justifies the increased application of radical novel and experimental treatment paradigms for all patients with glioblastoma.
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http://dx.doi.org/10.1016/j.mayocp.2018.11.031DOI Listing
July 2019

Potential influence of IDH1 mutation and MGMT gene promoter methylation on glioma-related preoperative seizures and postoperative seizure control.

Seizure 2019 Jul 20;69:283-289. Epub 2019 May 20.

Department of Pathology, Mayo Clinic, 4500 San Pablo Rd, Jacksonville, FL, 32224, United States. Electronic address:

Purpose: To examine the occurrence of glioma-related preoperative seizures (GPS) and post-operative seizure control (PSC) with respect to patients characteristics including five commonly tested tumor molecular markers (TMMs).

Methods: A single-center retrospective cohort study of patients with glioma evaluated at the Mayo Clinic, Florida between 2016 and 2018.

Results: 68 adult patients (mean age = 51-years, 45-males) were included. 46 patients had GPS. 57 patients underwent intra-operative electrocorticography during awake craniotomy-assisted glioma resection. All patients underwent glioma resection (53, gross-total resection) with histologies of pilocytic astrocytoma (n = 2), diffuse astrocytoma (n = 4), oligodendroglioma (n = 14), anaplastic astrocytoma (n = 16), anaplastic oligodendroglioma (n = 1), and glioblastoma (n = 31). 31 (67%) patients had PSC (median follow-up = 14.5 months; IQR = 7-16.5 months). IDH1 mutation (IDH1) was present in 32, ARTX retention in 53, MGMT gene promotor methylation in 15, 1p/19q co-deletion in 15, and over-expression of p53 in 19 patients. Patients with IDH1 were more likely to have GPS (p = 0.037) and PSC (p = 0.035) compared to patients with IDH1 wild-type. Patients with MGMT gene promoter methylation were also likely to have PSC (p = 0.032). GPS or PSC did not differ by age, sex, extent of surgery, glioma grade, location, and histopathological subtype, p53 expression, ARTX retention, or 1p/19q co-deletion status.

Conclusions: GPS and PSC may be associated with IDH1 mutation and MGMT gene promoter methylation status but not other glioma characteristics including tumor grade, location, or histopathology. Prospective studies with larger sample size are needed to clarify the exact mechanisms of GPS and PSC by the various TMMs to identify new treatment targets.
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http://dx.doi.org/10.1016/j.seizure.2019.05.018DOI Listing
July 2019

Survival trends in glioblastoma and association with treating facility volume.

J Clin Neurosci 2019 Oct 24;68:271-274. Epub 2019 May 24.

Department of Hematology and Oncology, Mayo Clinic, Jacksonville, FL, United States. Electronic address:

Glioblastoma (GBM) is one of the most lethal cancers. Various prognostic factors impact the survival of GBM patients. To further understand this extremely poor prognosis disease, we evaluated the effect of the treatment facility volumes on overall survival (OS) over the years, especially after the approval of multimodality therapy using temozolomide (TMZ) in 2005. National Cancer Data Base (NCDB) was utilized to identify GBM cases from 2004 through 2013 using ICD-O-3 code 9440/3 to identify eligible patients. We focused on studying the association between treatment facility volume and OS after adjusting for the patient-, disease-, and facility-characteristics. A total of 60,672 eligible GBM patients with median age of 65 years, treated at 1166 facilities were included in this analysis. The median annual facility volume was 3 patients/year (range: 0.1-55.1) and median OS was 8.1 months. There was an improvement in OS across all facilities after 2005, when multimodality therapy with TMZ was approved. Treatment at quartile 4 centers (Q4; >7 patients/year) was independently associated with decreased all-cause mortality in a multivariate analysis (Q3 hazard ratio [HR]: 1.11, 95% CI 1.09, 1.13; Q2 HR: 1.15, 95% CI 1.12, 1.19; Q1 HR: 1.25, 95% CI 1.17, 1.33). Treatment facility volume independently affects OS among GBM patients. Factors that are variable in high- and low-volume centers should be addressed to mitigate outcome disparities.
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http://dx.doi.org/10.1016/j.jocn.2019.04.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006067PMC
October 2019

A Brownian dynamics tumor progression simulator with application to glioblastoma.

Converg Sci Phys Oncol 2018 Mar 3;4(1). Epub 2018 Jan 3.

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, United States of America.

Tumor progression modeling offers the potential to predict tumor-spreading behavior to improve prognostic accuracy and guide therapy development. Common simulation methods include continuous reaction-diffusion (RD) approaches that capture mean spatio-temporal tumor spreading behavior and discrete agent-based (AB) approaches which capture individual cell events such as proliferation or migration. The brain cancer glioblastoma (GBM) is especially appropriate for such proliferation-migration modeling approaches because tumor cells seldom metastasize outside of the central nervous system and cells are both highly proliferative and migratory. In glioblastoma research, current RD estimates of proliferation and migration parameters are derived from computed tomography or magnetic resonance images. However, these estimates of glioblastoma cell migration rates, modeled as a diffusion coefficient, are approximately 1-2 orders of magnitude larger than single-cell measurements in animal models of this disease. To identify possible sources for this discrepancy, we evaluated the fundamental RD simulation assumptions that cells are point-like structures that can overlap. To give cells physical size (~10 m), we used a Brownian dynamics approach that simulates individual single-cell diffusive migration, growth, and proliferation activity via a gridless, off-lattice, AB method where cells can be prohibited from overlapping each other. We found that for realistic single-cell parameter growth and migration rates, a non-overlapping model gives rise to a jammed configuration in the center of the tumor and a biased outward diffusion of cells in the tumor periphery, creating a quasi-ballistic advancing tumor front. The simulations demonstrate that a fast-progressing tumor can result from minimally diffusive cells, but at a rate that is still dependent on single-cell diffusive migration rates. Thus, modeling with the assumption of physically-grounded volume conservation can account for the apparent discrepancy between estimated and measured diffusion of GBM cells and provide a new theoretical framework that naturally links single-cell growth and migration dynamics to tumor-level progression.
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http://dx.doi.org/10.1088/2057-1739/aa9e6eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322960PMC
March 2018

Microtubule-Based Control of Motor-Clutch System Mechanics in Glioma Cell Migration.

Cell Rep 2018 11;25(9):2591-2604.e8

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA; Physical Sciences-Oncology Center, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:

Microtubule-targeting agents (MTAs) are widely used chemotherapy drugs capable of disrupting microtubule-dependent cellular functions, such as division and migration. We show that two clinically approved MTAs, paclitaxel and vinblastine, each suppress stiffness-sensitive migration and polarization characteristic of human glioma cells on compliant hydrogels. MTAs influence microtubule dynamics and cell traction forces by nearly opposite mechanisms, the latter of which can be explained by a combination of changes in myosin motor and adhesion clutch number. Our results support a microtubule-dependent signaling-based model for controlling traction forces through a motor-clutch mechanism, rather than microtubules directly relieving tension within F-actin and adhesions. Computational simulations of cell migration suggest that increasing protrusion number also impairs stiffness-sensitive migration, consistent with experimental MTA effects. These results provide a theoretical basis for the role of microtubules and mechanisms of MTAs in controlling cell migration.
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http://dx.doi.org/10.1016/j.celrep.2018.10.101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345402PMC
November 2018

National care among patients with WHO grade I intracranial meningioma.

J Clin Neurosci 2018 Sep 15;55:17-24. Epub 2018 Jun 15.

Department of Radiation Oncology, Mayo Clinic, Jacksonville, FL, USA; Department of Neurological Surgery, Mayo Clinic, Jacksonville, FL, USA.

Purpose: To analyze the national treatment trends of patients diagnosed with benign intracranial meningioma.

Methods And Materials: Data was obtained from the National Cancer Database (NCDB) for patients with WHO grade I meningioma tumors between 2004 and 2014 (190,527 patients), diagnosed by either surgical specimen or diagnostic imaging. Univariable and multivariable analyses (binary logistic models) were performed to generate odds ratios (OR) and investigate factors associated with definitive initial treatment compared to initial observation. Initial treatments considered included surgical resection and/or radiation, including either fractionated external beam radiotherapy (EBRT) or stereotactic radiosurgery (SRS).

Results: The rate of observation increased over time, from 37% in 2004 to 55% in 2014 (p < 0.001). Conjointly, the rate of resection decreased from 50% to 37% from 2004 to 2014 (p < 0.001). The utilization of radiotherapy, including SRS, remained generally stable over time at 6% or less. SRS was more frequently utilized, compared to EBRT, as definitive treatment (4.6% versus 1.7%, respectively, p < 0.001). Compared to Community Cancer programs, patients at Academic/Research programs were more likely to receive definitive initial treatment over observation (OR = 2.909, each p < 0.001).

Conclusions: There is a national trend favoring initial observation for radiographically diagnosed WHO grade I meningioma. However, patients presenting to academic facilities are more likely to receive definitive initial treatment. Further research into differing approaches among treatment facilities for this common tumor may help clarify this trend.
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http://dx.doi.org/10.1016/j.jocn.2018.06.026DOI Listing
September 2018

High-frequency oscillations in awake patients undergoing brain tumor-related epilepsy surgery.

Neurology 2018 03 28;90(13):e1119-e1125. Epub 2018 Feb 28.

From the Departments of Neurology (A.M.F., W.O.T.), Neurological Surgery (D.M., K.R., A.Q.-H.), Hematology/Oncology (S.S.R.), and Anesthesiology (P.S.B.), Mayo Clinic, Jacksonville, FL; and Departments of Neurology (G.A.W., B.H.B.) and Physiology and Biomedical Engineering (G.A.W., B.H.B.), Mayo Clinic, Rochester, MN.

Objective: To examine the relationship between high-frequency oscillations (HFOs) and the presence of preoperative seizures, World Health Organization tumor grade, and isocitrate dehydrogenase 1 (IDH1) mutational status in gliomas.

Methods: We retrospectively studied intraoperative electrocorticography recorded in 16 patients with brain tumor (12 presenting with seizures) who underwent awake craniotomy and surgical resection between September 2016 and June 2017. The number and distribution of HFOs were determined and quantified visually and with an automated HFO detector.

Results: Five patients had low-grade (1 with grade I and 4 with grade II) and 11 had high-grade (6 with grade III and 5 with grade IV) brain tumors. An IDH1 mutation was found in 6 patients. Patients with a history of preoperative seizures were more likely to have HFOs than those without preoperative seizures (9 of 12 vs 0 of 4, = 0.02). The rate of HFOs was higher in patients with IDH1 mutant (mean 7.2 per minute) than IDH wild-type (mean 2.3 per minute) genotype ( = 0.03).

Conclusions: HFOs are common in brain tumor-related epilepsy, and HFO rate may be a useful measure of epileptogenicity in gliomas. Our findings further support the notion that IDH1 mutant genotype is more epileptogenic than IDH1 wild-type genotype gliomas.
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http://dx.doi.org/10.1212/WNL.0000000000005216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880636PMC
March 2018

A posttranslational modification of the mitotic kinesin Eg5 that enhances its mechanochemical coupling and alters its mitotic function.

Proc Natl Acad Sci U S A 2018 02 5;115(8):E1779-E1788. Epub 2018 Feb 5.

Department of Developmental and Cell Biology, University of California, Irvine, CA 92697.

Numerous posttranslational modifications have been described in kinesins, but their consequences on motor mechanics are largely unknown. We investigated one of these-acetylation of lysine 146 in Eg5-by creating an acetylation mimetic lysine to glutamine substitution (K146Q). Lysine 146 is located in the α2 helix of the motor domain, where it makes an ionic bond with aspartate 91 on the neighboring α1 helix. Molecular dynamics simulations predict that disrupting this bond enhances catalytic site-neck linker coupling. We tested this using structural kinetics and single-molecule mechanics and found that the K146Q mutation increases motor performance under load and coupling of the neck linker to catalytic site. These changes convert Eg5 from a motor that dissociates from the microtubule at low load into one that is more tightly coupled and dissociation resistant-features shared by kinesin 1. These features combined with the increased propensity to stall predict that the K146Q Eg5 acetylation mimetic should act in the cell as a "brake" that slows spindle pole separation, and we have confirmed this by expressing this modified motor in mitotically active cells. Thus, our results illustrate how a posttranslational modification of a kinesin can be used to fine tune motor behavior to meet specific physiological needs.
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http://dx.doi.org/10.1073/pnas.1718290115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828613PMC
February 2018

The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry.

Elife 2017 08 11;6. Epub 2017 Aug 11.

Institute of Structural and Molecular Biology, Birkbeck College, London, United Kingdom.

MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function.
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http://dx.doi.org/10.7554/eLife.27793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602324PMC
August 2017

Shifting the optimal stiffness for cell migration.

Nat Commun 2017 05 22;8:15313. Epub 2017 May 22.

Department of Biomedical Engineering, University of Minnesota, 312 Church Street SE, Minneapolis, Minnesota 55455, USA.

Cell migration, which is central to many biological processes including wound healing and cancer progression, is sensitive to environmental stiffness, and many cell types exhibit a stiffness optimum, at which migration is maximal. Here we present a cell migration simulator that predicts a stiffness optimum that can be shifted by altering the number of active molecular motors and clutches. This prediction is verified experimentally by comparing cell traction and F-actin retrograde flow for two cell types with differing amounts of active motors and clutches: embryonic chick forebrain neurons (ECFNs; optimum ∼1 kPa) and U251 glioma cells (optimum ∼100 kPa). In addition, the model predicts, and experiments confirm, that the stiffness optimum of U251 glioma cell migration, morphology and F-actin retrograde flow rate can be shifted to lower stiffness by simultaneous drug inhibition of myosin II motors and integrin-mediated adhesions.
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http://dx.doi.org/10.1038/ncomms15313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458120PMC
May 2017

Biphasic Dependence of Glioma Survival and Cell Migration on CD44 Expression Level.

Cell Rep 2017 01;18(1):23-31

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:

While several studies link the cell-surface marker CD44 to cancer progression, conflicting results show both positive and negative correlations with increased CD44 levels. Here, we demonstrate that the survival outcomes of genetically induced glioma-bearing mice and of high-grade human glioma patients are biphasically correlated with CD44 level, with the poorest outcomes occurring at intermediate levels. Furthermore, the high-CD44-expressing mesenchymal subtype exhibited a positive trend of survival with increased CD44 level. Mouse cell migration rates in ex vivo brain slice cultures were also biphasically associated with CD44 level, with maximal migration corresponding to minimal survival. Cell simulations suggest that cell-substrate adhesiveness is sufficient to explain this biphasic migration. More generally, these results highlight the potential importance of non-monotonic relationships between survival and biomarkers associated with cancer progression.
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http://dx.doi.org/10.1016/j.celrep.2016.12.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5498149PMC
January 2017

Matrix-driven Myosin II Mediates the Pro-fibrotic Fibroblast Phenotype.

J Biol Chem 2016 Mar 13;291(12):6083-95. Epub 2016 Jan 13.

From the Department of Pathobiology and Respiratory Institute,

Pro-fibrotic mesenchymal cells are known to be the key effector cells of fibroproliferative disease, but the specific matrix signals and the induced cellular responses that drive the fibrogenic phenotype remain to be elucidated. The key mediators of the fibroblast fibrogenic phenotype were characterized using a novel assay system that measures fibroblast behavior in response to actual normal and fibrotic lung tissue. Using this system, we demonstrate that normal lung promotes fibroblast motility and polarization, while fibrotic lung immobilizes the fibroblast and promotes myofibroblast differentiation. These context-specific phenotypes are surprisingly both mediated by myosin II. The role of myosin II is supported by the observation of an increase in myosin phosphorylation and a change in intracellular distribution in fibroblasts on fibrotic lung, as compared with normal lung. Moreover, loss of myosin II activity has opposing effects on protrusive activity in fibroblasts on normal and fibrotic lung. Loss of myosin II also selectively inhibits myofibroblast differentiation in fibroblasts on fibrotic lung. Importantly, these findings are recapitulated by varying the matrix stiffness of polyacrylamide gels in the range of normal and fibrotic lung tissue. Comparison of the effects of myosin inhibition on lung tissue with that of polyacrylamide gels suggests that matrix fiber organization drives the fibroblast phenotype under conditions of normal/soft lung, while matrix stiffness drives the phenotype under conditions of fibrotic/stiff lung. This work defines novel roles for myosin II as a key regulatory effector molecule of the pro-fibrotic phenotype, in response to biophysical properties of the matrix.
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http://dx.doi.org/10.1074/jbc.M115.712380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813589PMC
March 2016

The structural kinetics of switch-1 and the neck linker explain the functions of kinesin-1 and Eg5.

Proc Natl Acad Sci U S A 2015 Dec 16;112(48):E6606-13. Epub 2015 Nov 16.

Department of Cancer Biology, Lerner Research Institute of the Cleveland Clinic Foundation, Cleveland, OH 44195

Kinesins perform mechanical work to power a variety of cellular functions, from mitosis to organelle transport. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. Although crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction--including switch-1 and the neck linker--how movement of these two domains is coordinated through the ATPase cycle remains unknown. We have addressed this issue by using a novel combination of transient kinetics and time-resolved fluorescence, which we refer to as "structural kinetics," to map the timing of structural changes in the switch-1 loop and neck linker. We find that differences between the structural kinetics of Eg5 and kinesin-1 yield insights into how these two motors adapt their enzymologies for their distinct functions.
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http://dx.doi.org/10.1073/pnas.1512305112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672802PMC
December 2015

The mitotic kinesin KIF11 is a driver of invasion, proliferation, and self-renewal in glioblastoma.

Sci Transl Med 2015 Sep;7(304):304ra143

Department of Stem Cell Biology and Regenerative Medicine, Cleveland Clinic Foundation, Cleveland, OH 44195, USA. Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA.

The proliferative and invasive nature of malignant cancers drives lethality. In glioblastoma, these two processes are presumed mutually exclusive and hence termed "go or grow." We identified a molecular target that shuttles between these disparate cellular processes-the molecular motor KIF11. Inhibition of KIF11 with a highly specific small-molecule inhibitor stopped the growth of the more treatment-resistant glioblastoma tumor-initiating cells (TICs, or cancer stem cells) as well as non-TICs and impeded tumor initiation and self-renewal of the TIC population. Targeting KIF11 also hit the other arm of the "go or grow" cell fate decision by reducing glioma cell invasion. Administration of a KIF11 inhibitor to mice bearing orthotopic glioblastoma prolonged their survival. In its role as a shared molecular regulator of cell growth and motility across intratumoral heterogeneity, KIF11 is a compelling therapeutic target for glioblastoma.
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http://dx.doi.org/10.1126/scitranslmed.aac6762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743764PMC
September 2015

Multicenter phase 2 study of patupilone for recurrent or progressive brain metastases from non-small cell lung cancer.

Cancer 2015 Dec 26;121(23):4165-72. Epub 2015 Aug 26.

Center For Neuro-Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

Background: Treatment options for patients with non-small cell lung cancer (NSCLC) with brain metastases are limited. Patupilone (EPO906), a blood-brain barrier-penetrating, microtubule-targeting, cytotoxic agent, has shown clinical activity in phase 1/2 studies in patients with NSCLC. This study evaluates the efficacy, pharmacokinetics, and safety of patupilone in NSCLC brain metastases.

Methods: Adult patients with NSCLC and confirmed progressive brain metastases received patupilone intravenously at 10 mg/m(2) every 3 weeks. The primary endpoint of this multinomial 2-stage study combined early progression (EP; death or progression within 3 weeks) and progression-free survival at 9 weeks (PFS9w) to determine drug activity.

Results: Fifty patients with a median age of 60 years (range, 33-74 years) were enrolled; the majority were men (58%), and most had received prior therapy for brain metastases (98%). The PFS9w rate was 36%, and the EP rate was 26%. Patupilone blood pharmacokinetic analyses showed mean areas under the concentration-time curve from time zero to 504 hours for cycles 1 and 3 of 1544 and 1978 ng h/mL, respectively, and a mean steady state distribution volume of 755 L/m(2) . Grade 3/4 adverse events (AEs), regardless of their relation with the study drug, included diarrhea (24%), pulmonary embolisms (8%), convulsions (4%), and peripheral neuropathy (4%). All patients discontinued the study drug: 31 (62%) for disease progression and 13 (26%) for AEs. Twenty-five of 32 deaths were due to brain metastases. The median time to progression and the overall survival were 3.2 and 8.8 months, respectively.

Conclusions: This is the first prospective study of chemotherapy for recurrent brain metastases from NSCLC. In this population, patupilone demonstrated activity in heavily treated patients.
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http://dx.doi.org/10.1002/cncr.29636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5941922PMC
December 2015

Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins.

Elife 2014 Sep 10;3:e03680. Epub 2014 Sep 10.

Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, University of London, London, United Kingdom.

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles--including their nucleotide-free states--at ∼ 7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin-microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface.
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http://dx.doi.org/10.7554/eLife.03680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4358365PMC
September 2014

Trends in central nervous system tumor incidence relative to other common cancers in adults, adolescents, and children in the United States, 2000 to 2010.

Cancer 2015 Jan 25;121(1):102-12. Epub 2014 Aug 25.

Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio; Central Brain Tumor Registry of the United States (CBTRUS), Hinsdale, Illinois.

Background: Time trends in cancer incidence rates (IR) are important to measure the changing burden of cancer on a population over time. The overall IR of cancer in the United States is declining. Although central nervous system tumors (CNST) are rare, they contribute disproportionately to mortality and morbidity. In this analysis, the authors examined trends in the incidence of the most common cancers and CNST between 2000 and 2010.

Methods: The current analysis used data from the United States Cancer Statistics publication and the Central Brain Tumor Registry of the United States. Age-adjusted IR per 100,000 population with 95% confidence intervals and the annual percent change (APC) with 95% confidence intervals were calculated for selected common cancers and CNST overall and by age, sex, race/ethnicity, selected histologies, and malignancy status.

Results: In adults, there were significant decreases in colon (2000-2010: APC, -3.1), breast (2000-2010: APC, -0.8), lung (2000-2010: APC, -1.1), and prostate (2000-2010: APC, -2.4) cancer as well as malignant CNST (2008-2010: APC, -3.1), but a significant increase was noted in nonmalignant CNST (2004-2010: APC, 2.7). In adolescents, there were significant increases in malignant CNST (2000-2008: APC, 1.0) and nonmalignant CNST (2004-2010: APC, 3.9). In children, there were significant increases in acute lymphocytic leukemia (2000-2010: APC, 1.0), non-Hodgkin lymphoma (2000-2010: APC, 0.6), and malignant CNST (2000-2010: APC, 0.6).

Conclusions: Surveillance of IR trends is an important way to measure the changing public health and economic burden of cancer. In the current study, there were significant decreases noted in the incidence of adult cancer, whereas adolescent and childhood cancer IR were either stable or increasing.
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http://dx.doi.org/10.1002/cncr.29015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4298242PMC
January 2015

Comprehensive structural model of the mechanochemical cycle of a mitotic motor highlights molecular adaptations in the kinesin family.

Proc Natl Acad Sci U S A 2014 Feb 21;111(5):1837-42. Epub 2014 Jan 21.

Institute of Structural and Molecular Biology, Birkbeck College, London, WC1E 7HX, United Kingdon.

Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and essential cellular activities are poorly understood. It has been assumed that the first identified kinesin--the transport motor kinesin-1--is the mechanistic paradigm for the entire superfamily, but accumulating evidence suggests otherwise. To address the deficits in our understanding of the molecular basis of functional divergence within the kinesin superfamily, we studied kinesin-5s, which are essential mitotic motors whose inhibition blocks cell division. Using cryo-electron microscopy and determination of structure at subnanometer resolution, we have visualized conformations of microtubule-bound human kinesin-5 motor domain at successive steps in its ATPase cycle. After ATP hydrolysis, nucleotide-dependent conformational changes in the active site are allosterically propagated into rotations of the motor domain and uncurling of the drug-binding loop L5. In addition, the mechanical neck-linker element that is crucial for motor stepping undergoes discrete, ordered displacements. We also observed large reorientations of the motor N terminus that indicate its importance for kinesin-5 function through control of neck-linker conformation. A kinesin-5 mutant lacking this N terminus is enzymatically active, and ATP-dependent neck-linker movement and motility are defective, although not ablated. All these aspects of kinesin-5 mechanochemistry are distinct from kinesin-1. Our findings directly demonstrate the regulatory role of the kinesin-5 N terminus in collaboration with the motor's structured neck-linker and highlight the multiple adaptations within kinesin motor domains that tune their mechanochemistries according to distinct functional requirements.
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http://dx.doi.org/10.1073/pnas.1319848111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918802PMC
February 2014

Oculomotor nerve palsy as the presenting symptom of gummatous neurosyphilis and human immunodeficiency virus infection: clinical response to treatment.

JAMA Neurol 2013 Dec;70(12):1582-3

Department of Neurology, Neurological Institute of New York, Columbia University College of Physicians and Surgeons, New York, New York2University of Florida Center for Movement Disorders and Neurorestoration, Gainesville3Malcom Randall VA Medical Center, Gainesville, Florida.

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http://dx.doi.org/10.1001/jamaneurol.2013.1485DOI Listing
December 2013

Loop L5 assumes three distinct orientations during the ATPase cycle of the mitotic kinesin Eg5: a transient and time-resolved fluorescence study.

J Biol Chem 2013 Nov 21;288(48):34839-49. Epub 2013 Oct 21.

From the Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455.

Members of the kinesin superfamily of molecular motors differ in several key structural domains, which probably allows these molecular motors to serve the different physiologies required of them. One of the most variable of these is a stem-loop motif referred to as L5. This loop is longest in the mitotic kinesin Eg5, and previous structural studies have shown that it can assume different conformations in different nucleotide states. However, enzymatic domains often consist of a mixture of conformations whose distribution shifts in response to substrate binding or product release, and this information is not available from the "static" images that structural studies provide. We have addressed this issue in the case of Eg5 by attaching a fluorescent probe to L5 and examining its fluorescence, using both steady state and time-resolved methods. This reveals that L5 assumes an equilibrium mixture of three orientations that differ in their local environment and segmental mobility. Combining these studies with transient state kinetics demonstrates that there is a major shift in this distribution during transitions that interconvert weak and strong microtubule binding states. Finally, in conjunction with previous cryo-EM reconstructions of Eg5·microtubule complexes, these fluorescence studies suggest a model in which L5 regulates both nucleotide and microtubule binding through a set of reversible interactions with helix α3. We propose that these features facilitate the production of sustained opposing force by Eg5, which underlies its role in supporting formation of a bipolar spindle in mitosis.
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http://dx.doi.org/10.1074/jbc.M113.518845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3843096PMC
November 2013

Determinants of maximal force transmission in a motor-clutch model of cell traction in a compliant microenvironment.

Biophys J 2013 Aug;105(3):581-92

Department of Biomedical Engineering, University of Minnesota, Minneapolis, USA.

The mechanical stiffness of a cell's environment exerts a strong, but variable, influence on cell behavior and fate. For example, different cell types cultured on compliant substrates have opposite trends of cell migration and traction as a function of substrate stiffness. Here, we describe how a motor-clutch model of cell traction, which exhibits a maximum in traction force with respect to substrate stiffness, may provide a mechanistic basis for understanding how cells are tuned to sense the stiffness of specific microenvironments. We find that the optimal stiffness is generally more sensitive to clutch parameters than to motor parameters, but that single parameter changes are generally only effective over a small range of values. By contrast, dual parameter changes, such as coordinately increasing the numbers of both motors and clutches offer a larger dynamic range for tuning the optimum. The model exhibits distinct regimes: at high substrate stiffness, clutches quickly build force and fail (so-called frictional slippage), whereas at low substrate stiffness, clutches fail spontaneously before the motors can load the substrate appreciably (a second regime of frictional slippage). Between the two extremes, we find the maximum traction force, which occurs when the substrate load-and-fail cycle time equals the expected time for all clutches to bind. At this stiffness, clutches are used to their fullest extent, and motors are therefore resisted to their fullest extent. The analysis suggests that coordinate parameter shifts, such as increasing the numbers of motors and clutches, could underlie tumor progression and collective cell migration.
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http://dx.doi.org/10.1016/j.bpj.2013.06.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3736748PMC
August 2013
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