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On page 1 showing 1 ~ 20 papers out of 72 papers

Convulsive seizures from experimental focal cortical dysplasia occur independently of cell misplacement.

  • Lawrence S Hsieh‎ et al.
  • Nature communications‎
  • 2016‎

Focal cortical dysplasia (FCD), a local malformation of cortical development, is the most common cause of pharmacoresistant epilepsy associated with life-long neurocognitive impairments. It remains unclear whether neuronal misplacement is required for seizure activity. Here we show that dyslamination and white matter heterotopia are not necessary for seizure generation in a murine model of type II FCDs. These experimental FCDs generated by increasing mTOR activity in layer 2/3 neurons of the medial prefrontal cortex are associated with tonic-clonic seizures and a normal survival rate. Preventing all FCD-related defects, including neuronal misplacement and dysmorphogenesis, with rapamycin treatments from birth eliminates seizures, but seizures recur after rapamycin withdrawal. In addition, bypassing neuronal misplacement and heterotopia using inducible vectors do not prevent seizure occurrence. Collectively, data obtained using our new experimental FCD-associated epilepsy suggest that life-long treatment to reduce neuronal dysmorphogenesis is required to suppress seizures in individuals with FCD.


An epigenetic mechanism mediates developmental nicotine effects on neuronal structure and behavior.

  • Yonwoo Jung‎ et al.
  • Nature neuroscience‎
  • 2016‎

Developmental nicotine exposure causes persistent changes in cortical neuron morphology and in behavior. We used microarray screening to identify master transcriptional or epigenetic regulators mediating these effects of nicotine and discovered increases in Ash2l mRNA, encoding a component of a histone methyltransferase complex. We therefore examined genome-wide changes in trimethylation of histone H3 on Lys4 (H3K4me3), a mark induced by the Ash2l complex associated with increased gene transcription. A large proportion of regulated promoter sites were involved in synapse maintenance. We found that Mef2c interacts with Ash2l and mediates changes in H3K4me3. Knockdown of Ash2l or Mef2c abolished nicotine-mediated alterations of dendritic complexity in vitro and in vivo, and attenuated nicotine-dependent changes in passive avoidance behavior. In contrast, overexpression mimicked nicotine-mediated alterations of neuronal structure and passive avoidance behavior. These studies identify Ash2l as a target induced by nicotinic stimulation that couples developmental nicotine exposure to changes in brain epigenetic marks, neuronal structure and behavior.


Inhibition of p21 activated kinase (PAK) reduces airway responsiveness in vivo and in vitro in murine and human airways.

  • Wyn C Hoover‎ et al.
  • PloS one‎
  • 2012‎

The p21-activated protein kinases (Paks) have been implicated in the regulation of smooth muscle contractility, but the physiologic effects of Pak activation on airway reactivity in vivo are unknown. A mouse model with a genetic deletion of Pak1 (Pak1(-/-)) was used to determine the role of Pak in the response of the airways in vivo to challenge with inhaled or intravenous acetylcholine (ACh). Pulmonary resistance was measured in anesthetized mechanically ventilated Pak1(-/-) and wild type mice. Pak1(-/-) mice exhibited lower airway reactivity to ACh compared with wild type mice. Tracheal segments dissected from Pak1(-/-) mice and studied in vitro also exhibited reduced responsiveness to ACh compared with tracheas from wild type mice. Morphometric assessment and pulmonary function analysis revealed no differences in the structure of the airways or lung parenchyma, suggesting that that the reduced airway responsiveness did not result from structural abnormalities in the lungs or airways due to Pak1 deletion. Inhalation of the small molecule synthetic Pak1 inhibitor, IPA3, also significantly reduced in vivo airway responsiveness to ACh and 5-hydroxytryptamine (5-Ht) in wild type mice. IPA3 inhibited the contractility of isolated human bronchial tissues to ACh, confirming that this inhibitor is also effective in human airway smooth muscle tissue. The results demonstrate that Pak is a critical component of the contractile activation process in airway smooth muscle, and suggest that Pak inhibition could provide a novel strategy for reducing airway hyperresponsiveness.


Disruption of p21-activated kinase 1 gene diminishes atherosclerosis in apolipoprotein E-deficient mice.

  • Nikhlesh K Singh‎ et al.
  • Nature communications‎
  • 2015‎

Pak1 plays an important role in various cellular processes, including cell motility, polarity, survival and proliferation. To date, its role in atherogenesis has not been explored. Here we report the effect of Pak1 on atherogenesis using atherosclerosis-prone apolipoprotein E-deficient (ApoE(-/-)) mice as a model. Disruption of Pak1 in ApoE(-/-) mice results in reduced plaque burden, significantly attenuates circulating IL-6 and MCP-1 levels, limits the expression of adhesion molecules and diminishes the macrophage content in the aortic root of ApoE(-/-) mice. We also observed reduced oxidized LDL uptake and increased cholesterol efflux by macrophages and smooth muscle cells of ApoE(-/-):Pak1(-/-) mice as compared with ApoE(-/-) mice. In addition, we detect increased Pak1 phosphorylation in human atherosclerotic arteries, suggesting its role in human atherogenesis. Altogether, these results identify Pak1 as an important factor in the initiation and progression of atherogenesis.


A role of OCRL in clathrin-coated pit dynamics and uncoating revealed by studies of Lowe syndrome cells.

  • Ramiro Nández‎ et al.
  • eLife‎
  • 2014‎

Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.


PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

  • Hezhe Lu‎ et al.
  • Nature‎
  • 2017‎

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and β-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.


S phase entry of neural progenitor cells correlates with increased blood flow in the young subventricular zone.

  • Benjamin Lacar‎ et al.
  • PloS one‎
  • 2012‎

The postnatal subventricular zone (SVZ) contains proliferating neural progenitor cells in close proximity to blood vessels. Insults and drug treatments acutely stimulate cell proliferation in the SVZ, which was assessed by labeling cells entering S phase. Although G1-to-S progression is metabolically demanding on a minute-to-hour time scale, it remains unknown whether increased SVZ cell proliferation is accompanied by a local hemodynamic response. This neurovascular coupling provides energy substrates to active neuronal assemblies. Transcardial dye perfusion revealed the presence of capillaries throughout the SVZ that constrict upon applications of the thromboxane A(2) receptor agonist U-46119 in acute brain slice preparations. We then monitored in vivo blood flow using laser Doppler flowmetry via a microprobe located either in the SVZ or a mature network. U-46119 injections into the lateral ventricle decreased blood flow in the SVZ and the striatum, which are near the ventricle. A 1-hour ventricular injection of epidermal and basic fibroblast growth factor (EGF and bFGF) significantly increased the percentage of Sox2 transcription factor-positive cells in S phase 1.5 hours post-injection. This increase was accompanied by a sustained rise in blood flow in the SVZ but not in the striatum. Direct growth factor injections into the cortex did not alter local blood flow, ruling out direct effects on capillaries. These findings suggest that an acute increase in the number of G1-to-S cycling SVZ cells is accompanied by neurometabolic-vascular coupling, which may provide energy and nutrient for cell cycle progression.


p21-Activated kinases are required for transformation in a cell-based model of neurofibromatosis type 2.

  • Hoi Yee Chow‎ et al.
  • PloS one‎
  • 2010‎

NF2 is an autosomal dominant disease characterized by development of bilateral vestibular schwannomas and other benign tumors in central nervous system. Loss of the NF2 gene product, Merlin, leads to aberrant Schwann cell proliferation, motility, and survival, but the mechanisms by which this tumor suppressor functions remain unclear. One well-defined target of Merlin is the group I family of p21-activated kinases, which are allosterically inhibited by Merlin and which, when activated, stimulate cell cycle progression, motility, and increased survival. Here, we examine the effect of Pak inhibition on cells with diminished Merlin function.


Arpc1b, a centrosomal protein, is both an activator and substrate of Aurora A.

  • Poonam R Molli‎ et al.
  • The Journal of cell biology‎
  • 2010‎

Here we provide evidence in support of an inherent role for Arpc1b, a component of the Arp2/3 complex, in regulation of mitosis and demonstrate that its depletion inhibits Aurora A activation at the centrosome and impairs the ability of mammalian cells to enter mitosis. We discovered that Arpc1b colocalizes with gamma-tubulin at centrosomes and stimulates Aurora A activity. Aurora A phosphorylates Arpc1b on threonine 21, and expression of Arpc1b but not a nonphosphorylatable Arpc1b mutant in mammalian cells leads to Aurora A kinase activation and abnormal centrosome amplification in a Pak1-independent manner. Together, these findings reveal a new function for Arpc1b in centrosomal homeostasis. Arpc1b is both a physiological activator and substrate of Aurora A kinase and these interactions help to maintain mitotic integrity in mammalian cells.


Aspm knockout ferret reveals an evolutionary mechanism governing cerebral cortical size.

  • Matthew B Johnson‎ et al.
  • Nature‎
  • 2018‎

The human cerebral cortex is distinguished by its large size and abundant gyrification, or folding. However, the evolutionary mechanisms that drive cortical size and structure are unknown. Although genes that are essential for cortical developmental expansion have been identified from the genetics of human primary microcephaly (a disorder associated with reduced brain size and intellectual disability) 1 , studies of these genes in mice, which have a smooth cortex that is one thousand times smaller than the cortex of humans, have provided limited insight. Mutations in abnormal spindle-like microcephaly-associated (ASPM), the most common recessive microcephaly gene, reduce cortical volume by at least 50% in humans2-4, but have little effect on the brains of mice5-9; this probably reflects evolutionarily divergent functions of ASPM10,11. Here we used genome editing to create a germline knockout of Aspm in the ferret (Mustela putorius furo), a species with a larger, gyrified cortex and greater neural progenitor cell diversity12-14 than mice, and closer protein sequence homology to the human ASPM protein. Aspm knockout ferrets exhibit severe microcephaly (25-40% decreases in brain weight), reflecting reduced cortical surface area without significant change in cortical thickness, as has been found in human patients3,4, suggesting that loss of 'cortical units' has occurred. The cortex of fetal Aspm knockout ferrets displays a very large premature displacement of ventricular radial glial cells to the outer subventricular zone, where many resemble outer radial glia, a subtype of neural progenitor cells that are essentially absent in mice and have been implicated in cerebral cortical expansion in primates12-16. These data suggest an evolutionary mechanism by which ASPM regulates cortical expansion by controlling the affinity of ventricular radial glial cells for the ventricular surface, thus modulating the ratio of ventricular radial glial cells, the most undifferentiated cell type, to outer radial glia, a more differentiated progenitor.


Inhibition of MEK-ERK signaling reduces seizures in two mouse models of tuberous sclerosis complex.

  • Lena H Nguyen‎ et al.
  • Epilepsy research‎
  • 2022‎

Tuberous sclerosis complex (TSC) is a monogenic disorder characterized by hyperactivation of the mTOR signaling pathway and developmental brain malformations leading to intractable epilepsy. Although treatment with the recently approved mTOR inhibitor, everolimus, results in clinically relevant seizure suppression in up to 40% of TSC patients, seizures remain uncontrolled in a large number of cases, underscoring the need to identify novel treatment targets. The MEK-ERK signaling pathway has been found to be aberrantly activated in TSC and inhibition of MEK-ERK activity independently of mTOR rescued neuronal dendrite overgrowth in mice modeling TSC neuropathology. Here, we evaluated the efficacy of MEK-ERK inhibition on seizures in two mouse models of TSC. We found that treatment with the MEK inhibitor PD0325901 (mirdametinib) significantly reduced seizure activity in both TSC mouse models. These findings support inhibiting MEK-ERK activity as a potential alternative strategy to treat seizures in TSC.


Pak2 Regulation of Nrf2 Serves as a Novel Signaling Nexus Linking ER Stress Response and Oxidative Stress in the Heart.

  • Pablo Binder‎ et al.
  • Frontiers in cardiovascular medicine‎
  • 2022‎

Endoplasmic Reticulum (ER) stress and oxidative stress have been highly implicated in the pathogenesis of cardiac hypertrophy and heart failure (HF). However, the mechanisms involved in the interplay between these processes in the heart are not fully understood. The present study sought to determine a causative link between Pak2-dependent UPR activation and oxidative stress via Nrf2 regulation under pathological ER stress. We report that sustained ER stress and Pak2 deletion in cardiomyocytes enhance Nrf2 expression. Conversely, AAV9 mediated Pak2 delivery in the heart leads to a significant decrease in Nrf2 levels. Pak2 overexpression enhances the XBP1-Hrd1 UPR axis and ameliorates tunicamycin induced cardiac apoptosis and dysfunction in mice. We found that Pak2 deletion and altered proteostasis render Nrf2 detrimental by switching from its antioxidant role to renin-angiotensin aldosterone system (RAAS) gene regulator. Mechanistically, Pak2 mediated Hrd1 expression targets Nrf2 for ubiquitination and degradation thus preventing its aberrant activation. Moreover, we find a significant increase in Nrf2 with a decrease in Pak2 in human myocardium of dilated heart disease. Using human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we find that Pak2 is able to ameliorate Nrf2 induced RAAS activation under ER stress. These findings demonstrate that Pak2 is a novel Nrf2 regulator in the stressed heart. Activation of XBP1-Hrd1 is attributed to prevent ER stress-induced Nrf2 RAAS component upregulation. This mechanism explains the functional dichotomy of Nrf2 in the stressed heart. Thus, Pak2 regulation of Nrf2 homeostasis may present as a potential therapeutic route to alleviate detrimental ER stress and heart failure.


NetrinG1+ cancer-associated fibroblasts generate unique extracellular vesicles that support the survival of pancreatic cancer cells under nutritional stress.

  • Kristopher S Raghavan‎ et al.
  • Cancer research communications‎
  • 2022‎

It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEVs). The tumor-supportive CAFs possess a distinct phenotypic profile, compared to normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α5β1 localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1+ CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Further, we show that NetG1 expression in CAFs is required for the pro-survival properties of sEVs. Additionally, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α5β1 being enriched in exosomes. Finally, we found that NetG1 and Integrin α5β1 were detected in sEVs collected from plasma of PDAC patients, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor-stroma interactions and pathogenic stroma detection.


Limited View Tomographic Reconstruction Using a Cascaded Residual Dense Spatial-Channel Attention Network With Projection Data Fidelity Layer.

  • Bo Zhou‎ et al.
  • IEEE transactions on medical imaging‎
  • 2021‎

Limited view tomographic reconstruction aims to reconstruct a tomographic image from a limited number of projection views arising from sparse view or limited angle acquisitions that reduce radiation dose or shorten scanning time. However, such a reconstruction suffers from severe artifacts due to the incompleteness of sinogram. To derive quality reconstruction, previous methods use UNet-like neural architectures to directly predict the full view reconstruction from limited view data; but these methods leave the deep network architecture issue largely intact and cannot guarantee the consistency between the sinogram of the reconstructed image and the acquired sinogram, leading to a non-ideal reconstruction. In this work, we propose a cascaded residual dense spatial-channel attention network consisting of residual dense spatial-channel attention networks and projection data fidelity layers. We evaluate our methods on two datasets. Our experimental results on AAPM Low Dose CT Grand Challenge datasets demonstrate that our algorithm achieves a consistent and substantial improvement over the existing neural network methods on both limited angle reconstruction and sparse view reconstruction. In addition, our experimental results on Deep Lesion datasets demonstrate that our method is able to generate high-quality reconstruction for 8 major lesion types.


Molecular Imaging of Extracellular Tumor pH to Reveal Effects of Locoregional Therapy on Liver Cancer Microenvironment.

  • Lynn Jeanette Savic‎ et al.
  • Clinical cancer research : an official journal of the American Association for Cancer Research‎
  • 2020‎

To establish magnetic resonance (MR)-based molecular imaging paradigms for the noninvasive monitoring of extracellular pH (pHe) as a functional surrogate biomarker for metabolic changes induced by locoregional therapy of liver cancer.


Extracellular 5'-methylthioadenosine inhibits intracellular symmetric dimethylarginine protein methylation of FUSE-binding proteins.

  • Baiqing Tang‎ et al.
  • The Journal of biological chemistry‎
  • 2022‎

Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway that converts the polyamine synthesis byproduct 5'-deoxy-5'-methylthioadenosine (MTA) into methionine. Inactivation of MTAP, often by homozygous deletion, is found in both solid and hematologic malignancies and is one of the most frequently observed genetic alterations in human cancer. Previous work established that MTAP-deleted cells accumulate MTA and contain decreased amounts of proteins with symmetric dimethylarginine (sDMA). These findings led to the hypothesis that accumulation of intracellular MTA inhibits the protein arginine methylase (PRMT5) responsible for bulk protein sDMAylation. Here, we confirm that MTAP-deleted cells have increased MTA accumulation and reduced protein sDMAylation. However, we also show that addition of extracellular MTA can cause a dramatic reduction of the steady-state levels of sDMA-containing proteins in MTAP+ cells, even though no sustained increase in intracellular MTA is found because of catabolism of MTA by MTAP. We determined that inhibition of protein sDMAylation by MTA occurs within 48 h, is reversible, and is specific. In addition, we have identified two enhancer-binding proteins, FUBP1 and FUBP3, that are differentially sDMAylated in response to MTAP and MTA. These proteins work via the far upstream element site located upstream of Myc and other promoters. Using a transcription reporter construct containing the far upstream element site, we demonstrate that MTA addition can reduce transcription, suggesting that the reduction in FUBP1 and FUBP3 sDMAylation has functional consequences. Overall, our findings show that extracellular MTA can inhibit protein sDMAylation and that this inhibition can affect FUBP function.


Semi-natural housing rescues social behavior and reduces repetitive exploratory behavior of BTBR autistic-like mice.

  • Matthew S Binder‎ et al.
  • Scientific reports‎
  • 2023‎

Environmental enrichment confers numerous benefits when implemented in murine models and can reduce behavioral symptomatology in models of disease, such as autism spectrum disorder (ASD). However, previous work did not examine the impact of early-life environmental enrichment on each core feature of ASD. We thus implemented a social and physical enrichment at birth, modeling a semi-natural housing, and examined its impact on communicative, social, sensory, and repetitive behaviors using BTBR (autistic-like) and C57BL/6 J (B6, wildtype) mice, comparing them to standard housing conditions. We found that environmental enrichment alleviated the social deficit of juvenile BTBR mice and reduced their repetitive exploratory behavior but did not affect their rough versus smooth texture preference nor the number of maternal isolation-induced pup calls. Environmental enrichment only affected the call characteristics of B6 mice. One interpretation of these data is that early-life environmental enrichment has significant therapeutic potential to treat selective core features of ASD. Another interpretation is that reducing environmental complexity causes selective behavioral deficits in ASD-prone mice suggesting that current standard housing may be suboptimal. Overall, our data illustrate the extent to which the environment influences behavior and highlights the importance of considering housing conditions when designing experiments and interpreting behavioral results.


The mTOR pathway genes MTOR, Rheb, Depdc5, Pten, and Tsc1 have convergent and divergent impacts on cortical neuron development and function.

  • Lena H Nguyen‎ et al.
  • eLife‎
  • 2024‎

Brain somatic mutations in various components of the mTOR complex 1 (mTORC1) pathway have emerged as major causes of focal malformations of cortical development and intractable epilepsy. While these distinct gene mutations converge on excessive mTORC1 signaling and lead to common clinical manifestations, it remains unclear whether they cause similar cellular and synaptic disruptions underlying cortical network hyperexcitability. Here, we show that in utero activation of the mTORC1 activator genes, Rheb or MTOR, or biallelic inactivation of the mTORC1 repressor genes, Depdc5, Tsc1, or Pten in the mouse medial prefrontal cortex leads to shared alterations in pyramidal neuron morphology, positioning, and membrane excitability but different changes in excitatory synaptic transmission. Our findings suggest that, despite converging on mTORC1 signaling, mutations in different mTORC1 pathway genes differentially impact cortical excitatory synaptic activity, which may confer gene-specific mechanisms of hyperexcitability and responses to therapeutic intervention.


PAK kinase regulates Rac GTPase and is a potential target in human schwannomas.

  • Christine Flaiz‎ et al.
  • Experimental neurology‎
  • 2009‎

Merlin loss causes benign tumours of the nervous system, mainly schwannomas and meningiomas. Schwannomas show enhanced Rac1 and Cdc42 activity, the p21-activated kinase 2 (PAK2) activation and increased ruffling and cell adhesion. PAK regulates activation of merlin. PAK has been proposed as a potential therapeutic target in schwannomas. However where PAK stands in the Rac pathway is insufficiently characterised. We used a novel small-molecule PAK inhibitor, IPA-3, to investigate the role of PAK activation on Rac1/Cdc42 activity, cell spreading and adhesion in human primary schwannoma and Schwann cells. We show that IPA-3 blocks activation of PAK2 at Ser192/197 that antagonises PAK's interaction with Pix. Accordingly, Pix-mediated Rac1 activation is decreased in IPA-3 treated schwannoma cells, indicating that PAK acts upstream of Rac. We show that this Rac activation at the level of focal adhesions in schwannoma cells is essential for cell spreading and adhesion in Schwann and schwannoma cells.


Application of multiplexed kinase inhibitor beads to study kinome adaptations in drug-resistant leukemia.

  • Matthew J Cooper‎ et al.
  • PloS one‎
  • 2013‎

Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.


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