Pyk2 Signaling Research Articles

Selective Pyk2 inhibition enhances bone restoration through SCARA5-mediated bone marrow remodeling in ovariectomized mice

Understanding the intricate cellular interactions involved in bone restoration is crucial for developing effective strategies to promote bone healing and mitigate conditions such as osteoporosis and fractures. Here, we provide compelling evidence supporting the anabolic effects of a pharmacological Pyk2 inhibitor (Pyk2-Inh) in promoting bone restoration. In vitro, Pyk2 signaling inhibition markedly enhances alkaline phosphatase (ALP) activity, a hallmark of osteoblast differentiation, through activation of canonical Wnt/β-catenin signaling. Notably, analysis of human mesenchymal stem cells through RNA-seq revealed a novel candidate, SCARA5, identified through Pyk2-Inh treatment. We demonstrate that Scara5 plays a crucial role in suppressing the differentiation from stromal cells into adipocytes, and accelerates lineage commitment to osteoblasts, establishing Scara5 as a negative regulator of bone formation. Additionally, Pyk2 inhibition significantly impedes osteoclast differentiation and bone resorption. In a co-culture system comprising osteoblasts and osteoclasts, Pyk2-Inh effectively suppressed osteoclast differentiation, accompanied by a substantial increase in the transcriptional expression of Tnfrsf11b and Csf1 in osteoblasts, highlighting a dual regulatory role in osteoblast-osteoclast crosstalk. In an ovariectomized mouse model of osteoporosis, oral administration of Pyk2-Inh significantly increased bone mass by simultaneously reducing bone resorption, promoting bone formation and decreasing bone marrow fat. These results suggest Pyk2 as a potential therapeutic target for both adipogenesis and osteogenesis in bone marrow. Our findings underscore the importance of Pyk2 signaling inhibition as a key regulator of bone remodeling, offering promising prospects for the development of novel osteoporosis therapies.

Enhanced phagocytosis associated with multinucleated microglia via Pyk2 inhibition in an acute β-amyloid infusion model

Multinucleated microglia have been observed in contexts associated with infection, inflammation, and aging. Though commonly linked to pathological conditions, the larger cell size of multinucleated microglia might enhance their phagocytic functions, potentially aiding in the clearance of brain debris and suggesting a reassessment of their pathological significance. To assess the phagocytic capacity of multinucleated microglia and its implications for brain debris clearance, we induced their formation by inhibiting Pyk2 activity using the pharmacological inhibitor PF-431396, which triggers cytokinesis regression. Multinucleated microglia demonstrate enhanced phagocytic function, as evidenced by their increased capacity to engulf β-amyloid (Aβ) oligomers. Concurrently, the phosphorylation of Pyk2, induced by Aβ peptide, was diminished upon treatment with a Pyk2 inhibitor (Pyk2-Inh, PF-431396). Furthermore, the increased expression of Lamp1, a lysosomal marker, with Pyk2-inh treatment, suggests an enhancement in proteolytic activity. In vivo, we generated an acute Alzheimer’s disease (AD) model by infusing Aβ into the brains of Iba-1 EGFP transgenic (Tg) mice. The administration of the Pyk2-Inh led to an increased migration of microglia toward amyloid deposits in the brains of Iba-1 EGFP Tg mice, accompanied by morphological activation, suggesting a heightened affinity for Aβ. In human microglia, lipopolysaccharide (LPS)-induced inflammatory responses showed that inhibition of Pyk2 signaling significantly reduced the transcription and protein expression of pro-inflammatory markers. These results suggest that Pyk2 inhibition can modulate microglial functions, potentially reducing neuroinflammation and aiding in the clearance of neurodegenerative disease markers. This highlights Pyk2 as a promising target for therapeutic intervention in neurodegenerative diseases.

Abstract 5564: The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling

Abstract Glioblastoma (GBM), the most aggressive brain cancer which is usually fatal within a year after diagnosis, poses a significant therapeutic challenge with conventional treatments providing limited success. Despite advancements in immune checkpoint blockade, PD-L1 inhibitors exhibit suboptimal efficacy in recurrent GBMs with recent clinical trials demonstrating only 8% objective responses in patients. Utilizing the R2 Genomic Analysis Visualization Platform, we identified a positive 8-18% correlation between Pyk2 and PD-L1 gene expression in human GBM specimens. We hypothesize that Pyk2 signaling is involved in the regulation of PD-L1 expression in GBM cells. In this study, primary human glioblastoma cell lines and GL261 mouse glioma cells were assessed employing CRISPR/Cas9 Pyk2 knockout (Pyk2KO) combined with pharmacological Pyk2 inhibition. Flow cytometric analysis revealed a 35% decrease in PD-L1 expression in Pyk2KO GL261 cells compared to wild-type cells. SiRNA knockdown against Pyk2 in primary human GBM cells demonstrated a similar response, with a 50% decrease in PD-L1 expression compared to MOCK control cells. Additionally, the application of the Pyk2/FAK inhibitor defactinib at a concentration of 0.2 μM reduced PD-L1 expression by 32% in both GL261 and primary human GBM cells. These findings highlight Pyk2 as a potential therapeutic target for modulating the immunosuppressive microenvironment in GBM and enhancing the effectiveness of immune checkpoint blockade therapies. This study was supported by: NIH Grant 1SC1GM122691 and PRSTRT2022. Citation Format: Tyrel Porter, Lilia Kucheryavykh. The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5564.

Molecularly imprinted nanoparticles reveal regulatory scaffolding features in Pyk2 tyrosine kinase.

Pyk2 is a multi-domain non-receptor tyrosine kinase that serves dual roles as a signaling enzyme and scaffold. Pyk2 activation involves a multi-stage cascade of conformational rearrangements and protein interactions initiated by autophosphorylation of a linker site. Linker phosphorylation recruits Src kinase, and Src-mediated phosphorylation of the Pyk2 activation loop confers full activation. The regulation and accessibility of the initial Pyk2 autophosphorylation site remains unclear. We employed peptide-binding molecularly imprinted nanoparticles (MINPs) to probe the regulatory conformations controlling Pyk2 activation. MINPs differentiating local structure and phosphorylation state revealed that the Pyk2 autophosphorylation site is protected in the autoinhibited state. Activity profiling of Pyk2 variants implicated FERM and linker residues responsible for constraining the autophosphorylation site. MINPs targeting each Src docking site disrupt the higher-order kinase interactions critical for activation complex maturation. Ultimately, MINPs targeting key regulatory motifs establish a useful toolkit for probing successive activational stages in the higher-order Pyk2 signaling complex.

TMIC-51. THE RELEASE OF INFLAMMATORY CYTOKINES ARE REGULATED BY PYK2 SIGNALING IN GBM CELLS

Abstract Glioblastoma (GBM) is a highly aggressive brain cancer that is typically fatal within a year of diagnosis. Previous research has shown a positive relationship between the activation of a protein called proline-rich non-receptor tyrosine kinase (Pyk2) in GBM cells in addition to the expression of cytokines by tumor-infiltrating myeloid (TIM cells). Leading to a sustained increase in the expression levels of monocyte chemoattractant protein 1 (CCL2), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL10, IL12, and vascular endothelial growth factor (VEGF), as well as an increase in the CD86+/CD206+ TIM population. We hypothesize that Pyk2 signaling in GBM cells is involved in the regulation of the release of inflammatory cytokines, which in turn suppresses the immune response in the tumor microenvironment. We used CRISPR/Cas9 knockout Pyk2 (Pyk2KO) and wild type (Pyk2WT) GL261 mouse glioma cells in this study. Analysis of the cytokines present in the cell culture medium conditioned by Pyk2KO and Pyk2WT cells identified a decrease in the release of CCL2 and CCL5 in the Pyk2KO cells compared to the Pyk2WT cells. RT-PCR analysis of myeloid cell population, isolated from Pyk2WT and Pyk2KO tumors in the GL261-C57Bl/6 GBM model, identified a significant down-regulation of key pro-tumorigenic factors such as CCL2, CCL12, CCL5, tumor necrosis factor (TNF), VEGF and epidermal growth factor (EGF) in the Pyk2KO tumors compared to the Pyk2WT tumors. Additionally, flow cytometry analysis revealed a reduction in myeloid-derived suppressor cells (MDSCs), an increase in lymphoid dendritic cells (DC), and up-regulation in TNF/IFNg expressing and antigen-specific CD8+ T cells in Pyk2KO tumors. Furthermore, a significant decrease in pro-tumorigenic Ly6C+/CD206+ and an up-regulation of inflammatory Ly6C+/CD86+ myeloid cells were found in the Pyk2KO tumors. These findings suggest that Pyk2KO tumors display an immunocompetent microenvironment with enhanced phagocytic and cytotoxic function compared to Pyk2WT tumors. NIH Grant 1SC1GM122691 and PRSTRT2022 supported this study

The PYK2 inhibitor PF-562271 enhances the effect of temozolomide on tumor growth in a C57Bl/6-Gl261 mouse glioma model

BackgroundThe development of resistance to temozolomide (TMZ), a standard chemotherapeutic, limits the effective treatment of glioblastoma (GBM). Focal adhesion kinase (FAK) and proline rich tyrosine kinase 2 (Pyk2) regulate proliferation and invasion of GBM cells. We found that TMZ activates FAK and Pyk2 signaling in GBM. We hypothesized that pharmacological inhibitors of Pyk2/FAK together with TMZ can enhance the inhibitory effect of TMZ on tumor growth and dispersal and improve the treatment outcome.MethodsPrimary human GBM cell cultures and a C57Bl/6-GL261 mouse glioma implantation model were used. Pyk2 (Tyr579/580) and FAK (Tyr925) phosphorylation was analyzed by western blotting. Viability, cell cycle, migration, invasion and invadopodia formation were investigated in vitro. Animal survival, tumor size and invasion, TUNEL apoptotic cell death and the Ki67 proliferation index were evaluated in vivo upon treatment with TMZ (50 mg/kg, once/day, orally) and the Pyk2/FAK inhibitor PF-562271 (once/daily, 50 mg/kg, orally) vs. TMZ monotherapy.ResultsIn vitro studies revealed significantly reduced viability, cell cycle progression, invasion and invadopodia with TMZ (100 µM) + PF-562271 (16 nM) compared with TMZ alone. In vivo studies demonstrated that combinatorial treatment led to prominent reductions in tumor size and invasive margins, extensive signs of apoptosis and a reduced proliferation index, together with a 15% increase in the survival rate in animals, compared with TMZ monotherapy.ConclusionTMZ + PF-562271 eliminates TMZ-related Pyk2/FAK activation in GBM and improves the treatment efficacy.

TMIC-07. PYK2 SIGNALING IS UPREGULATED IN RECURRENT GLIOBLASTOMA TUMORS IN C57BL/6/GL261 GLIOMA IMPLANTATION MODEL

Abstract Glioblastoma (GBM) is the most aggressive type of brain cancer with no successful treatment available. Microglia makes up to 30% of total GBM tumor mass. Previously we demonstrated that microglia within the tumor stimulate glioma cell growth and invasion through the proline rich tyrosine kinase 2 (Pyk2). Tumor resection is the first line treatment for GBM. Our previous studies with use of mouse glioma implantation model indicate that microglia in a site of tumor resection undergo significant modulations in cytokines expression profile. We hypothesize that microglia activated by tumor resection induces Pyk2 signaling in glioma cell and promotes malignant properties of the tumor cells that fail to be eliminated by resection. Using C57BL/6/GL261 mouse glioma implantation model, we analyzed primary implanted tumors and re-grown tumor after surgical resection. Two weeks after glioma cells implantation, tumor resection was performed, and two weeks later regrown tumors were used for western blot analysis. Total Pyk2 and phosphorylated Pyk2[Y579/50] were analyzed. The study revealed that Pyk2 phosphorylation is upregulated in re-grown tumor compared to primary implanted tumor. Treatment with PF-562271 (Pyk2/FAK inhibitor, once/daily, 50mg/kg, orally) beginning the 2ndday after tumor resection, reduced Pyk2 phosphorylation in regrown tumors compared to animals that did not receive treatment with PF-562271. In conclusion we can state that tumor resection procedure induces the upregulation of Pyk2 phosphorylation in regrown tumors. This effect is eliminated with oral administration of PF-562271.

Quanzhenyiqitang Reverses LPS-Induced Inflammation via Inhibiting PYK2/p38MAPK/HDAC2/CK2 Signaling Pathway in Rat Alveolar Macrophage.

Chronic obstructive pulmonary disease (COPD) is a common chronic pulmonary disease with multiple etiologies and pathological changes. PYK2 expression is significantly increased in lipopolysaccharide-induced lung injury, but it mediates chronic lung inflammation. The mechanism of its occurrence remains unclear. Quanzhenyiqitang is often used in clinical treatment of COPD, so this study explored the mechanism of its treatment of lipopolysaccharide-induced lung injury. In this study, transfection, flow cytometry, QRT-PCR, and Western blotting methods were used to study the mechanism of Quanzhenyiqitang lipopolysaccharide-induced lung injury. The results showed that the mechanism of occurrence remains unclear. Our novel observations imply that the PYK2/p38MAPK/HDAC2/CK2 pathway is one of the fundamental underlying mechanisms that mediate the pathogenic progression of COPD, and Quanzhenyiqitang may be the therapeutic drug to prevent chronic inflammation and delay the progression of COPD by inhibiting PYK2 signaling pathways.

Microglial Cytokines Induce Invasiveness and Proliferation of Human Glioblastoma through Pyk2 and FAK Activation

Simple SummaryMicroglia infiltrate most gliomas and have been demonstrated to promote tumor growth, invasion, and treatment resistance. To develop improved treatment methods, that take into consideration the supporting role of microglia in tumor progression, the functional and mechanistic pathways of glioma–microglia interactions need to be identified and experimentally dissected. Our recent studies and literature reports revealed the overexpression of Pyk2 and FAK in glioblastomas. Pyk2 and FAK signaling pathways have been shown to regulate migration and proliferation in glioma cells, including microglia-promoted glioma cell migration. However, the specific factors released by microglia that modulate Pyk2 and FAK to promote glioma invasiveness and proliferation are poorly understood. The aim of this study was to identify key microglia-derived signaling molecules that induce the activation of Pyk2- and FAK-dependent glioma cell proliferation and invasiveness.Glioblastoma is the most aggressive brain tumor in adults. Multiple lines of evidence suggest that microglia create a microenvironment favoring glioma invasion and proliferation. Our previous studies and literature reports indicated the involvement of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) in glioma cell proliferation and invasion, stimulated by tumor-infiltrating microglia. However, the specific microglia-released factors that modulate Pyk2 and FAK signaling in glioma cells are unknown. In this study, 20 human glioblastoma specimens were evaluated with the use of RT-PCR and western blotting. A Pierson correlation test demonstrated a correlation (0.6–1.0) between the gene expression levels for platelet-derived growth factor β(PDGFβ), stromal-derived factor 1α (SDF-1α), IL-6, IL-8, and epidermal growth factor (EGF) in tumor-purified microglia and levels of p-Pyk2 (Y579/Y580) and p-FAK(Y925) in glioma cells. siRNA knockdown against Pyk2 or FAK in three primary glioblastoma cell lines, developed from the investigated specimens, in combination with the cytokine receptor inhibitors gefitinib (1 μM), DMPQ (200 nM), and burixafor (1 μM) identified EGF, PDGFβ, and SDF-1α as key extracellular factors in the Pyk2- and FAK-dependent activation of invadopodia formation and the migration of glioma cells. EGF and IL-6 were identified as regulators of the Pyk2- and FAK-dependent activation of cell viability and mitosis.

TAMI-45. MICROGLIAL CYTOKINES INDUCE INVASIVENESS AND PROLIFERATION THROUGH PYK2 AND FAK ACTIVATION IN HUMAN GLIOBLASTOMA CELL

Abstract Glioblastoma (GBM) is the most aggressive and highly invasive primary brain tumor in adults. Evidence suggests that microglia create a microenvironment favoring glioma invasion and proliferation. Indeed, previous reports indicate the involvement of focal adhesion kinase (FAK) signaling cascades in glioma cell proliferation. Besides, studies from our laboratory support a critical role of Pyk2, a relative of FAK, in glioma invasion by tumor-infiltrating microglia. However, the microglial-released factors modulating Pyk2 and FAK signaling pathways are unknown. In this study, 20 human GBM specimens were evaluated to identify the cytokine expression patterns in purified microglia and FAK and Pyk2 phosphorylation in glioma cell fraction by RT-PCR and western blot. A Pierson correlation test demonstrated a high correlation (0.8-1.0) of gene expression for PDGFα, PDGFβ, SDF-1α, IL-6, IL-8, and EGF in percoll-purified microglia, and pPyk2(Y579/580) and pFAK(Y925) levels in glioma cell fraction. The role of cytokines in cell invasion and proliferation by Pyk2/FAK activation was further investigated in primary cell lines from three patients. Thirty percent up-regulation of pPyk2 and pFAK was detected in glioma cells treated (2 hrs.) with microglia conditioned media (MCM) compared to control cells. siPyk2 or siFAK knockdown identified IL-6 (100 μM) and EGF (1 μM) as key factors of Pyk2- and FAK-dependent activation in all glioma cell lines. Similar results with siPyk2 or siFAK were observed for matrix degradation, invadopodia formation, cell viability, and mitosis. Indeed, Tocilizumab (IL-6R blocker, 100 ng/mL) and Gefitinib (EGFR blocker, 1 μM) reversed the effect of MCM on glioma cell proliferation and invasion in all cell lines evaluated. These findings support a pivotal role of Pyk2 and FAK in enhancing proliferation and invasion of glioma tumors through IL-6 and EGF-dependent pathways. The latter could be of clinical relevance for new therapeutic developments in GBM patients.

A PKCβ-LYN-PYK2 Signaling Axis Is Critical for MCP-1-Dependent Migration and Adhesion of Monocytes.

MCP-1-induced monocyte chemotaxis is a crucial event in inflammation and atherogenesis. Identifying the important signal transduction pathways that control monocyte chemotaxis can unravel potential targets for preventive therapies in inflammatory disease conditions. Previous studies have shown that the focal adhesion kinase Pyk2 plays a critical role in monocyte motility. In this study, we investigated the MCP-1-mediated activation of Pyk2 (particularly by the phosphorylation of Tyr402) in primary human peripheral blood monocytes. We showed that MCP-1 induces Src phosphorylation in a similar time frame and that the MCP-1-induced Pyk2 tyrosine phosphorylation is controlled by the Src family kinase. We also report, in this study, that PKCβ, an isoform of PKC, is required for both Src and Pyk2 activation/phosphorylation in response to MCP-1 stimulation. We identified Lyn as the specific Src kinase isoform that is activated by MCP-1 and acts upstream of Pyk2 in primary monocytes. Furthermore, Lyn is found to be indispensable for monocyte migration in response to MCP-1 stimulation. Moreover, our coimmunoprecipitation studies in monocytes revealed that PKCβ, Pyk2, and Lyn exist constitutively in a molecular complex. To our knowledge, our study has uncovered a novel PKCβ-Lyn-Pyk2 signaling cascade in primary monocytes that regulates MCP-1-induced monocyte adhesion and migration.

Orai1 Channel Inhibition Preserves Left Ventricular Systolic Function and Normal Ca2+ Handling After Pressure Overload.

Orai1 is a critical ion channel subunit, best recognized as a mediator of store-operated Ca2+ entry (SOCE) in nonexcitable cells. SOCE has recently emerged as a key contributor of cardiac hypertrophy and heart failure but the relevance of Orai1 is still unclear. To test the role of these Orai1 channels in the cardiac pathophysiology, a transgenic mouse was generated with cardiomyocyte-specific expression of an ion pore-disruptive Orai1R91W mutant (C-dnO1). Synthetic chemistry and channel screening strategies were used to develop 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline (hereafter referred to as JPIII), a small-molecule Orai1 channel inhibitor suitable for in vivo delivery. Adult mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and reduced ventricular function associated with increased Orai1 expression and Orai1-dependent SOCE (assessed by Mn2+ influx). C-dnO1 mice displayed normal cardiac electromechanical function and cellular excitation-contraction coupling despite reduced Orai1-dependent SOCE. Five weeks after TAC, C-dnO1 mice were protected from systolic dysfunction (assessed by preserved left ventricular fractional shortening and ejection fraction) even if increased cardiac mass and prohypertrophic markers induction were observed. This is correlated with a protection from TAC-induced cellular Ca2+ signaling alterations (increased SOCE, decreased [Ca2+]i transients amplitude and decay rate, lower SR Ca2+ load and depressed cellular contractility) and SERCA2a downregulation in ventricular cardiomyocytes from C-dnO1 mice, associated with blunted Pyk2 signaling. There was also less fibrosis in heart sections from C-dnO1 mice after TAC. Moreover, 3 weeks treatment with JPIII following 5 weeks of TAC confirmed the translational relevance of an Orai1 inhibition strategy during hypertrophic insult. The findings suggest a key role of cardiac Orai1 channels and the potential for Orai1 channel inhibitors as inotropic therapies for maintaining contractility reserve after hypertrophic stress.

EGFR signaling pathway occupies an important position in cancer-related downstream signaling pathways of Pyk2.

Proline‐rich tyrosine kinase 2 (Pyk2) is a member of focal adhesion kinase (FAK) non‐receptor tyrosine kinase family and has been found to promote cancer cell survival, proliferation, migration, invasion, and metastasis. Pyk2 takes part in different carcinogenic signaling pathways to promote cancer progression, including epidermal growth factor receptor (EGFR) signaling pathway. EGFR signaling pathway is a traditional carcinogenic signaling pathway, which plays a critical role in tumorigenesis and tumor progression. FAK inhibitors have been reported to fail to get the ideal anti‐cancer outcomes because of activation of EGFR signaling pathway. Better understanding of Pyk2 downstream targets and interconnectivity between Pyk2 and carcinogenic EGFR signaling pathway will help finding more effective targets for clinical anti‐cancer combination therapies. Thus, the interconnectivity between Pyk2 and EGFR signaling pathway, which regulates tumor development and metastasis, needs to be elucidated. In this review, we summarized the downstream targets of Pyk2 in cancers, focused on the connection between Pyk2 and EGFR signaling pathway in different cancer types, and provided a new overview of the roles of Pyk2 in EGFR signaling pathway and cancer development.

Inhibition of didscoidin domain receptor 1 reduces epithelial-mesenchymal transition and induce cell-cycle arrest and apoptosis in prostate cancer cell lines.

Didscoidin domain receptor 1 (DDR1) is involved in the progression of prostate cancer metastasis through stimulation of epithelial-mesenchymal transition (EMT). So DDR1 inhibition can be a helpful target for cancer metastasis prevention. So, we studied the effects of DDR1 inhibition on EMT as well as induction of cell-cycle arrest and apoptosis in prostate cancer cell lines. DDR1 expression was evaluated using reverse-transcription polymerase chain reaction and western blot analysis. The EMT-associated protein expression was determined using the western blot analysis and immunocytochemistry following treatment with various concentrations of DDR1 inhibitor. The activation of DDR1 and also downstream-signaling molecules Pyk2 and MKK7 were determined using western blot analysis. Cell survival and proliferation after DDR1 inhibition were evaluated using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide, bromodeoxyuridine, and colony formation assays. Flow cytometry analysis was used to determine the effects of DDR1 inhibition on cell-cycle arrest and apoptosis using annexin V/propidium iodide-based flow cytometry. Results showed that the protein expression of N-cadherin and vimentin were decreased whereas protein expression of E-cadherin was increased after DDR1 inhibition. Results of our western blot analysis indicated that DDR1 inhibitor effectively downregulated P-DDR1, P-Pyk2, and P-MKK7 levels. This result also showed that DDR1 inhibition decreased cell survival and proliferation, induced G1 cell-cycle arrest, induced apoptosis by an increase in the Bax/Bcl-2 ratio and depletion of the mitochondrial membrane potential, and also by reactive oxygen species creation in prostate cancer cells. These data show that DDR1 inhibition can result in the EMT prevention via inhibition of Pyk2 and MKK7 signaling pathway and induces cell-cycle arrest and apoptosis in prostate cancer cell lines. Thus, this study identifies DDR1 as an important target for modulating EMT and induction of apoptosis in prostate cancer cells.

Pyk2 Signaling through Graf1 and RhoA GTPase Is Required for Amyloid-β Oligomer-Triggered Synapse Loss.

The intracellular tyrosine kinase Pyk2 (PTK2B) is related to focal adhesion kinase and localizes to postsynaptic sites in brain. Pyk2 genetic variation contributes to late onset Alzheimer's disease (AD) risk. We recently observed that Pyk2 is required for synapse loss and for learning deficits in a transgenic mouse model of AD. Here, we explore the cellular and biochemical basis for the action of Pyk2 tyrosine kinase in amyloid-β oligomer (Aβo)-induced dendritic spine loss. Overexpression of Pyk2 reduces dendritic spine density of hippocampal neurons by a kinase-dependent mechanism. Biochemical isolation of Pyk2-interacting proteins from brain identifies Graf1c, a RhoA GTPase-activating protein inhibited by Pyk2. Aβo-induced reductions in dendritic spine motility and chronic spine loss require both Pyk2 kinase and RhoA activation. Thus, Pyk2 functions at postsynaptic sites to modulate F-actin control by RhoA and regulate synapse maintenance of relevance to AD risk.SIGNIFICANCE STATEMENT Genetic variation at the Pyk2 locus is a risk for Alzheimer's disease. We have observed that Pyk2 is required for AD transgenic synapse loss and memory dysfunction. However, the cellular and biochemical basis for Pyk2 function related to AD is not defined. Here, we show that brain Pyk2 interacts with the RhoGAP protein Graf1 to alter dendritic spine stability via RhoA GTPase. Amyloid-β oligomer-induced dendritic spine loss requires the Pyk2/Graf1 pathway.

Abstract 1092: PF562271, a Pyk2 inhibitor, reduces glioma tumor growth and invasion in C57Bl6-Gl261 mouse glioma model

Abstract Glioblastoma is an extraordinarily aggressive type of brain cancer due to its invasive and proliferative nature. The tumor microenvironment, with microglia as a critical player, has an important role in tumor progression. Microglia infiltrate majority of gliomas and release factors, which favor tumor growth and invasion. Previously we demonstrated that microglia residing within the tumor stimulate glioma cell invasion through the proline rich tyrosine kinase 2 (Pyk2) signaling cascade. We hypothesize that the use of pharmacological inhibitors of Pyk2 together with currently applied chemotherapy can significantly reduce invasiveness of glioblastoma tumor and improve the outcome of the treatment. Using C57Bl/6-Gl261 mouse glioma implantation model we investigated the effect of the combined treatment for glioblastoma with temozolomide (TMZ, 50 mg/kg, once/day, orally) together with a Pyk2 inhibitor PF562271 (twice/daily, 25 mg/kg, orally) vs. TMZ monotherapy. Treatment was provided during 14 days, beginning the 5th day after glioma implantation. For the assessment of the effectiveness of treatment animals’ survival, tumor size, and invasion area were evaluated. Western blot were used for the evaluation of the level of Pyk2 phosphorylation in tumor cells. For the purification of glioma cells from total tumor tissue Percol gradients were used. The study revealed that treatment with PF562271 reduced invasion of glioma cells at the tumor edge, while TMZ reduced the tumor growth. Combined treatment showed significantly more prominent effect on reduction of tumors growth compared to TMZ monotherapy and additionally reduced invasiveness of tumors similar to PF562271 monotherapy. In both PF562271 monotherapy and combined treatments the downregulation of Pyk2 phosphorylation in glioma cells has been recorded. Survival analysis demonstrated a significance increase of survival of animals received combined treatment compare to TMZ monotherapy. In conclusion, we can state that combined treatment with TMZ together with PF562271 reduced Pyk2-related tumor growth and invasiveness and increased animal survival. This research was made possible by NIH grant numbers: 1SC2GM102040, G12MD007583, R25GM110513, Puerto Rico Science, Technology, and Research Trust grant number 2016-00157, and US Department of Education Grant number P031S130068. Citation Format: Jescelica Ortiz-Rivera, Kimberleve Rolón-Reyes, Alejandro Albors, Luis Cubano, Lilia Kucheryavykh. PF562271, a Pyk2 inhibitor, reduces glioma tumor growth and invasion in C57Bl6-Gl261 mouse glioma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1092. doi:10.1158/1538-7445.AM2017-1092

Sperm-oocyte contact induces outside-in signaling via PYK2 activation

Fertilization is a multi-step process that begins with plasma membrane interactions that enable sperm – oocyte binding followed by fusion of the sperm and oocyte plasma membranes. Once membrane fusion has occurred, sperm incorporation involves actin remodeling events within the oocyte cortex that allow the sperm head to penetrate the cortical actin layer and gain access to the ooplasm. Despite the significance for reproduction, the control mechanisms involved in gamete binding, fusion, and sperm incorporation are poorly understood. While it is known that proline – rich tyrosine kinase 2 (PYK2 or PTK2b) kinase activity plays an important role in fertilization, its specific function has not been addressed. The present study made use of a zona-free mouse oocyte fertilization assay to investigate the relationship between PYK2 activity and sperm – oocyte binding and fusion, as well as localized changes in actin polymerization and sperm incorporation. In this assay, the majority of bound sperm had no apparent effect on the oocyte and only a few became incorporated into the ooplasm. However, a subset of bound sperm were associated with a localized response in which PYK2 was recruited to the oocyte cortex where it frequently co-localized with a ring or disk of f-actin. The frequency of sperm-oocyte binding sites that exhibited this actin response was reduced in pyk2-/- oocytes and the pyk2-/- oocytes proved less efficient at incorporating sperm, indicating that this protein kinase may have an important role in sperm incorporation. The response of PYK2 to sperm-oocyte interaction appeared unrelated to gamete fusion since PYK2 was recruited to sperm – binding sites under conditions where sperm – oocyte fusion was prevented and since PYK2 suppression or ablation did not prevent sperm – oocyte fusion. While a direct correlation between the PYK2 response in the oocyte and the successful incorporation of individual bound sperm remains to be established, these findings suggest a model in which the oocyte is not a passive participant in fertilization, but instead responds to sperm contact by localized PYK2 signaling that promotes actin remodeling events required to physically incorporate the sperm head into the ooplasm.

Periodic Mechanical Stress INDUCES Chondrocyte Proliferation and Matrix Synthesis via CaMKII-Mediated Pyk2 Signaling

Background/Aims: Periodic mechanical stress can promote chondrocyte proliferation and matrix synthesis to improve the quality of tissue-engineered cartilage. Although the integrin β1–ERK1/2 signal cascade has been implicated in periodic mechanical stress-induced mitogenic effects in chondrocytes, the precise mechanisms have not been fully established. The current study was designed to probe the roles of CaMKII and Pyk2 signaling in periodic mechanical stress-mediated chondrocyte proliferation and matrix synthesis. Methods: Chondrocytes were subjected to periodic mechanical stress, proliferation was assessed by direct cell counting and CCK-8 assay; gene expressions were analyzed using quantitative real-time PCR, protein abundance by Western blotting. Results: Mechanical stress, markedly enhanced the phosphorylation levels of Pyk2 at Tyr⁴⁰² and CaMKII at Thr²⁸⁶. Both suppression of Pyk2 with Pyk2 inhibitor PF431396 or Pyk2 shRNA and suppression of CaMKII with CaMKII inhibitor KN-93 or CaMKII shRNA blocked periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. Additionally, either pretreatment with KN-93 or shRNA targeted to CaMKII prevented the activation of ERK1/2 and Pyk2 under conditions of periodic mechanical stress. Interestingly, in relation to periodic mechanical stress, in the context of Pyk2 inhibition with PF431396 or its targeted shRNA, only the phosphorylation levels of ERK1/2 were abrogated, while CaMKII signal activation was not affected. Moreover, the phosphorylation levels of CaMKII- Thr²⁸⁶ and Pyk2- Tyr⁴⁰² were abolished after pretreatment with blocking antibody against integrinβ1 exposed to periodic mechanical stress. Conclusion: Our results collectively indicate that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis through the integrinβ1–CaMKII–Pyk2–ERK1/2 signaling cascade.

Abstract B202: Combined therapy of temozolomide and PF-562271, a PYK2 inhibitor, reduces glioma tumor growth and dispersal compare to temozolomide monotherapy

Abstract Microglia infiltrate majority of gliomas and release factors, which favor tumor growth and dispersal. Previously we demonstrated that microglia residing within the tumor stimulate glioma cell dispersal through the proline rich tyrosine kinase 2 (Pyk2) signaling cascade (Rolon-Reyes et al. 2015). We hypothesize that the use of pharmacological inhibitors of Pyk2 in applied therapy can significantly reduce the dispersal of tumor cells and improve the outcome of treatment. In the present study, we investigated both in vitro and in vivo, the effect of the combined treatment of glioblastoma with temozolomide (TMZ) together with PF-562271 (a Pyk2 and FAK inhibitor), vs. TMZ monotherapy. Proliferation and migration assays were performed for a number of primary human glioma cell lines, as well as U87 and HS683cell lines in the presence and absence of microglia. Tumor size and invasion area were evaluated in brains of GL261 tumor bearing C57BL/6 mice for the assessment of the effectiveness of treatment. Our data demonstrate that PF-562271 (16nM) and TMZ (100μM) given alone reduced in vitro migration and proliferation of glioma cells in the presence of microglia. PF-562271 (16nM) given in combination with TMZ at a concentration of just 10μM provided the same effect as TMZ 100 μM along, without reduction of efficacy. In vivo experiments demonstrated that oral administration of PF-562271 (twice/daily, 25 mg/kg, orally) reduced invasion of glioma cells at the tumor edge, while TMZ (50 mg/kg, once/day, orally) reduced the tumor growth. Combined treatment revealed significantly more prominent effect on both tumor growth and invasion compare to monotherapy. These data indicate that the combination of TMZ and PF-562271 reduces both tumor growth and dispersal of tumor cells to surrounding areas. Moreover, in vitro experiments revealed a complex effect of these drugs on glioma cell proliferation and migration, allowing the reduction of the applied concentration of TMZ without the loss of its therapeutic effect. This research was made possible by NIH grant numbers: 1SC2GM102040-01A1, 8G12MD007583-27, Title V PPOHA grant number P031M105050, NIH NIMH R25 MH055929 and RO1-CA18382701A1. Citation Format: Kimberleve Rolon-Reyes, Luis A. Cubano, Alfredo Quiñones-Hinojosa, Lilia Kucheryavykh. Combined therapy of temozolomide and PF-562271, a PYK2 inhibitor, reduces glioma tumor growth and dispersal compare to temozolomide monotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B202.

Anti-inflammatory activities of phospholipase C inhibitor U73122: Inhibition of monocyte-to-macrophage transformation and LPS-induced pro-inflammatory cytokine expression

A wide range of biological processes are controlled by phospholipase C (PLC)/Ca(2+) signaling, which could be blocked by PLC-specific inhibitor U73122. Whether inhibition of PLC with chemical inhibitor U73122 affects the inflammatory response in monocytes/macrophages is currently unknown. In this study, we demonstrated that U73122 inhibited PMA-induced in vitro differentiation of human promonocytic U937 cells into macrophages as reflected by the reduction of cell adherence and the decreased expression of macrophage specific marker CD163. It is possible that U73122 blocked PMA-induced adhesion of U937 cells partially by down regulation and inactivation of both Pyk2 and paxillin signaling. Furthermore, the expression of LPS-induced pro-inflammatory cytokines TNF-α and IL-1β was significantly blocked by U73122 in both dU937 cells and mouse primary peritoneal macrophages. These results suggest that PLC is involved in the sophisticated inflammatory response by monocytes/macrophages, and thereby chemical antagonists of PLC may be potential agents for the suppression of inflammatory response.