Characterization of Common Genetic Variants in P2RX7 and Their Contribution to Chronic Pain Conditions.

The adenosine triphosphate (ATP)-gated channel P2X7 is encoded by a gene enriched for common nonsynonymous variants. Many of these variants have functional cellular effects, and some have been implicated in chronic pain. In this study, we first systematically characterized all 17 common nonsynonymous variants using whole-cell patch clamp electrophysiology. Then, we analyzed these variants for statistical association with chronic pain phenotypes using both individual P2RX7 variants as predictors and cumulative allele counts of same-direction cellular effect in univariate models. Association and validation analyses were conducted in the Orofacial Pain: Prospective Evaluation and Risk Assessment (OPPERA) cohort (N = 3260) and in the Complex Persistent Pain Conditions (CPPC) cohort (N = 900), respectively. Our results showed an association between allele A of rs7958311 and an increased risk of chronic pelvic pain, with convergent evidence for contribution to fibromyalgia and irritable bowel syndrome, confirmed in a meta-analysis. This allelic variant produced a unique cellular phenotype: a gain-of-function in channel opening, and a loss-of-function in pore opening. A computational study using a 12-state Markov model of ATP binding to the P2X7 receptor suggested that this cellular phenotype arises from an increased ATP binding affinity and an increased open channel conductance combined with a loss of sensitization. Cumulative allele count analysis did not provide additional insights. In conclusion, our results go beyond reproducing association for rs7958311 with chronic pain and suggest that its unique combination of gain-of-function in channel and loss-of-function in pore activity may explain why it is likely the only common P2RX7 variant with contribution to chronic pain. PERSPECTIVE: This study characterizes all common P2RX7 variants using cellular assays and statistical association analyses with chronic pain, with Markov state modeling of the most robustly associated variant.

Mechanosensitivity and mechanotransductive properties of osteoclasts.

Mechanical loading is essential for maintaining bone health. Here, we aimed to investigate the role of ATP and ADP in the mechanotransduction of bone-resorptive osteoclasts. Single osteoclast in primary cultures from 10 to 12-wk-old mice was mechanically stimulated by a gentle touch with a micropipette. Changes in cytosolic free calcium [Ca2+]i were analyzed in Fura-2 loaded osteoclasts. The cell injury was assessed by analyzing the cellular Fura-2 loss and classified as severe or mild using k-means. Osteoclasts responded to mechanical stimuli with transient calcium elevation (primary responders) and transduced these signals to neighboring cells, which responded with delayed calcium elevations (secondary responders). Severely injured osteoclasts had higher calcium transients than mildly injured cells. Fluid shear stress similarly induced reversible cell injury in osteoclasts. Secondary responses were abolished by treatment with A-804598, a specific inhibitor of P2X7, but not suramin, a broad P2 receptor blocker. Osteoclasts responded to ATP and ADP with concentration-dependent changes in [Ca2+]i. We performed osteoclast micropipette stimulation in the presence of phosphoenolpyruvate and pyruvate kinase which converted all ADP in solution to ATP, or with hexokinase converting all ATP to ADP. Osteoclasts with mild membrane injury demonstrated similar calcium responses in ATP and ADP-rich environments. However, when the mechanotransductive signal to severe osteoclast injury was converted to ADP, the fraction of secondary responders and their [Ca2+]i amplitude was higher. This study suggests the importance of osteoclast mechanobiology and the role of ADP-mediated signaling in conditions of altered mechanical loading associated with bone loss.NEW & NOTEWORTHY Osteoclasts are rarely considered as cells that participate in mechanical signaling in bone. We show that osteoclasts are capable of sensing and transmitting mechanical signals to neighboring cells. Mechanical stimulation commonly induces minor repairable membrane injury in osteoclasts. ATP and especially ADP were found to play important roles in the mechanoresponsiveness of osteoclasts. This study highlights the importance of osteoclast mechanobiology especially in conditions of altered mechanical loading associated with bone loss, such as in microgravity.

Efficacy of antiresorptive agents in fibrous dysplasia and McCune Albright syndrome, a systematic review and meta-analysis.

Fibrous dysplasia (FD) is a rare skeletal disorder in which normal bone is replaced by a fibro-osseous tissue, resulting in possible deformities and fractures. The aim of this systematic review and meta-analysis was to synthesize the available evidence on the use of antiresorptive drugs in FD in terms of changes in bone turnover markers (BTMs), bone mineral density (BMD), and reducing pain. Three databases were searched in October 2022, with an update in July 2023. Of the 1037 studies identified, 21 were retained after eligibility assessment. A random-effects model was used to calculate global effect size and the corresponding standard error. Pamidronate and Denosumab were the most reported drugs in a total of 374 patients assessed. The initiation of treatments was accompanied by an average reduction of 40.5% [CI95% -51.6, -29.3] in the bone resorption parameters, and 22.0% [CI95% -31.9, -12.1] in the parameters of bone formation after 6-12 months. BMD was increased in both FD lesions and in the unaffected skeleton. Pain was reduced by 32.7% [CI95% -52.7, -12.6] after 6-12 months of treatment, and by 44.5% [CI95% -65.3, -23.6] after a mean 41.2 months of follow-up. The variation in pain was highly correlated to variation in bone resorption (R2 = 0.08, p < 0.0001) and formation parameters (R2 = 0.17, p < 0.0001). This study supports the overall efficacy of antiresorptive therapies in terms of reducing bone remodeling, improving bone density, and pain in FD.

Epidemiology and Genotype Distribution of Hepatitis C Virus in Russia.

The hepatitis C virus (HCV) causes both acute and chronic infection of the liver that can lead to liver cirrhosis, cancer, and liver failure. HCV is characterized by high genetic diversity and substantial variations in the prevalence of specific HCV genotypes throughout the world. Many effective regimens of direct-acting antivirals (DAAs), including pan-genotypic, can successfully treat HCV infection. Additionally, genotype-specific treatments for HCV are being actively employed in national plans for eliminating HCV infection around the world. The evaluation of HCV genotype prevalence in a given country is necessary for the successful implementation of the HCV elimination plans and for allocating financial resources to the DAAs which are the most effective against those specific HCV genotypes prevalent in a given country. Here, we analyzed HCV genotypes, subgenotypes, and recombinants in 10,107 serum samples collected in 2015-2017 from patients with chronic HCV infection living in all federal districts of Russia. This is the first and largest evaluation of HCV genotypes performed on samples from all territories of Russia, from its Central federal district to the Far East. Moreover, we have updated retrospective epidemiological analysis of chronic and acute HCV infection in Russia from 2001 to 2021. We demonstrate that the incidence of acute HCV (AHC) infection in Russia decreased from 16.7 cases per 100,000 people in 2001 to 0.6/100,000 in 2021. The number of cases of chronic HCV (CHC) infection also decreased from 29.5 to 16.4 per 100,000 people during this period. The HCV genotype analysis indicated that HCV genotype 1 dominates in Russia (53.6%), while genotypes 3 and 2 were detected in 35.4% and 7.8% of patients, respectively. These proportions are virtually identical in all regions of Russia except for the Far East, where HCV genotype 2 was detected in only 1% of the samples. HCV genotypes 1 and 2 are more widespread in women, and HCV genotype 3 in men. Genotype 3 was the most prevalent in 31-40-year-olds (44.9%), and genotype 1 was most prevalent in those over 70 years of age (72.2%). HCV genotype 2 was predominant among HCV-infected persons older than 40 years. Discriminating between HCV genotype 2 and recombinant RF1_2k/1b, which are frequently misclassified, is important for successful antiviral treatment. For the first time, we demonstrate, here, countrywide prevalence of HCV RF1_2k/1b in different regions of Russia. HCV RF1_2k/1b makes up 3.2% of HCV genotypes, reaching 30% among samples classified as genotype 2 by some commercial genotyping tests. The highest proportion of HCV RF1_2k/1b was detected in the North-West (60%), Southern (41.6%), and Central (31.6%) federal districts; its frequency in the Far Eastern and North Caucasus districts was ~14.3%. HCV RF1_2k/1b, and it was not detected in the Volga, Ural, or Siberian districts. To conclude, this is the first and most complete evaluation of HCV epidemiology and genotype/subgenotype distribution in Russia.

Extracellular ATP and its derivatives provide spatiotemporal guidance for bone adaptation to wide spectrum of physical forces.

ATP is a ubiquitous intracellular molecule critical for cellular bioenergetics. ATP is released in response to mechanical stimulation through vesicular release, small tears in cellular plasma membranes, or when cells are destroyed by traumatic forces. Extracellular ATP is degraded by ecto-ATPases to form ADP and eventually adenosine. ATP, ADP, and adenosine signal through purinergic receptors, including seven P2X ATP-gated cation channels, seven G-protein coupled P2Y receptors responsive to ATP and ADP, and four P1 receptors stimulated by adenosine. The goal of this review is to build a conceptual model of the role of different components of this complex system in coordinating cellular responses that are appropriate to the degree of mechanical stimulation, cell proximity to the location of mechanical injury, and time from the event. We propose that route and amount of ATP release depend on the scale of mechanical forces, ranging from vesicular release of small ATP boluses upon membrane deformation, to leakage of ATP through resealable plasma membrane tears, to spillage of cellular content due to destructive forces. Correspondingly, different P2 receptors responsive to ATP will be activated according to their affinity at the site of mechanical stimulation. ATP is a small molecule that readily diffuses through the environment, bringing the signal to the surrounding cells. ATP is also degraded to ADP which can stimulate a distinct set of P2 receptors. We propose that depending on the magnitude of mechanical forces and distance from the site of their application, ATP/ADP profiles will be different, allowing the relay of information about tissue level injury and proximity. Lastly, ADP is degraded to adenosine acting via its P1 receptors. The presence of large amounts of adenosine without ATP, indicates that an active source of ATP release is no longer present, initiating the transition to the recovery phase. This model consolidates the knowledge regarding the individual components of the purinergic system into a conceptual framework of choreographed responses to physical forces.

Platelets and osteoblasts: secretome connections.

Megakaryocyte hyperplasia associated with myeloproliferative neoplasms commonly leads to abnormal bone tissue deposition in the bone marrow, known as osteosclerosis. In this study, we aimed to synthesize the known proteomics literature describing factors released by megakaryocytes and platelets and to examine if any of the secreted factors have a known ability to stimulate the bone-forming cells, osteoblasts. Using a systematic search of Medline, we identified 77 articles reporting on factors secreted by platelets and megakaryocytes. After a full-text screening and analysis of the studies, we selected seven papers that reported proteomics data for factors secreted by platelets from healthy individuals. From 60 proteins reported in at least two studies, we focused on 23 that contained a putative signal peptide, which we searched for a potential osteoblast-stimulatory function. From nine proteins with a positive effect on osteoblast formation and function, two extracellular matrix (ECM) proteins, secreted protein acidic and rich in cysteine (SPARC) and tissue inhibitor of metalloproteinase-1 (TIMP1), and three cellular proteins with known extracellular function, the 70-kDa heat shock protein (HSP70), thymosin-ß4 (TB4), and super dismutase (SOD), were identified as hypothetical candidate molecules to be examined as potential mediators in mouse models of osteomyelofibrosis. Thus, careful analysis of prior literature can be beneficial in assisting the planning of future experimental studies.

Megakaryocyte-bone cell interactions: lessons from mouse models of experimental myelofibrosis and related disorders.

Over the years, numerous studies demonstrated reciprocal communications between processes of bone marrow hematopoiesis and bone remodeling. Megakaryocytes, rare bone marrow cells responsible for platelet production, were demonstrated to be involved in bone homeostasis. Myelofibrosis, characterized by an increase in pleomorphic megakaryocytes in the bone marrow, commonly leads to the development of osteosclerosis. In vivo, an increase in megakaryocyte number was shown to result in osteosclerosis in GATA-1low, Nf-e2-/-, TPOhigh, Mplf/f;PF4cre, Lnk-/-, Mpig6b-/-, Mpig6bfl/fl;Gp1ba-Cr+/KI, and Pt-vWD mouse models. In vitro, megakaryocytes stimulate osteoblast proliferation and have variable effects on osteoclast proliferation and activity through soluble factors and direct cell-cell communications. Intriguingly, new studies revealed that the ability of megakaryocytes to communicate with bone cells is affected by the age and sex of animals. This mini-review summarizes changes seen in bone architecture and bone cell function in mouse models with an elevated number of megakaryocytes and the effects megakaryocytes have on osteoblasts and osteoclasts in vitro, and discusses potential molecular players that can mediate these effects.

High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts.

The P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consists of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca2+ responses to ATP, including a transition of Ca2+ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10-4 and 10-2 M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP3) mediated Ca2+ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca2+ responses due to the biphasic nature of IP3Rs and the interaction of SERCA pumps with IP3Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP3Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanotransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca2+ response signatures, which are important in mediating bone mechanoadaptation.

Characterization of Potency of the P2Y13 Receptor Agonists: A Meta-Analysis.

P2Y13 is an ADP-stimulated G-protein coupled receptor implicated in many physiological processes, including neurotransmission, metabolism, pain, and bone homeostasis. Quantitative understanding of P2Y13 activation dynamics is important for translational studies. We systematically identified PubMed annotated studies that characterized concentration-dependence of P2Y13 responses to natural and synthetic agonists. Since the comparison of the efficacy (maximum response) is difficult for studies performed in different systems, we normalized the data and conducted a meta-analysis of EC50 (concentration at half-maximum response) and Hill coefficient (slope) of P2Y13-mediated responses to different agonists. For signaling events induced by heterologously expressed P2Y13, EC50 of ADP-like agonists was 17.2 nM (95% CI: 7.7-38.5), with Hills coefficient of 4.4 (95% CI: 3.3-5.4), while ATP-like agonists had EC50 of 0.45 µM (95% CI: 0.06-3.15). For functional responses of endogenously expressed P2Y13, EC50 of ADP-like agonists was 1.76 µM (95% CI: 0.3-10.06). The EC50 of ADP-like agonists was lower for the brain P2Y13 than the blood P2Y13. ADP-like agonists were also more potent for human P2Y13 compared to rodent P2Y13. Thus, P2Y13 appears to be the most ADP-sensitive receptor characterized to date. The detailed understanding of tissue- and species-related differences in the P2Y13 response to ADP will improve the selectivity and specificity of future pharmacological compounds.

Active hematopoiesis triggers exosomal release of PRDX2 that promotes osteoclast formation.

Hematopoietic disorders, particularly hemolytic anemias, commonly lead to bone loss. We have previously reported that actively proliferating cancer cells stimulate osteoclastogenesis from late precursors in a RANKL-independent manner. We theorized that cancer cells exploit the physiological role of bone resorption to support expanding hematopoietic bone marrow and examined if hematopoietic cells can trigger osteoclastogenesis. Using phlebotomy-induced acute anemia in mice, we found strong correlation between augmented erythropoiesis and increased osteoclastogenesis. Conditioned medium (CM) from K562 erythroleukemia cells and primary mouse erythroblasts stimulated osteoclastogenesis when added to RANKL-primed precursors from mouse bone marrow or RAW264.7 cells. Using immunoblotting and mass spectrometry, PRDX2 was identified as a factor produced by erythroid cells in vitro and in vivo. PRDX2 was detected in K562-derived exosomes, and inhibiting exosomal release significantly decreased the osteoclastogenic capacity of K562 CM. Recombinant PRDX2 induced osteoclast formation from RANKL-primed primary or RAW 264.7 precursors to levels comparable to achieved with continuous RANKL treatment. Thus, increased bone marrow erythropoiesis secondary to anemia leads to upregulation of PRDX2, which is released in the exosomes and acts to induce osteoclast formation. Increased bone resorption by the osteoclasts expands bone marrow cavity, which likely plays a supporting role to increase blood cell production.

Severity of Megakaryocyte-Driven Osteosclerosis in Mpig6b-Deficient Mice Is Sex-Linked.

Patients with chronic myelofibrosis often suffer from osteosclerosis, which is associated with bone pain and may lead to bone marrow failure. The pathogenesis of myelofibrosis is linked to aberrant megakaryocyte development and function. Null and loss-of-function mutations in MPIG6B, which codes for the inhibitory heparan sulfate receptor G6b-B, result in severe macrothrombocytopenia, large megakaryocyte clusters, and focal primary myelofibrosis in mice and humans. We investigated the development of osteosclerosis in Mpig6b null (Mpig6b-/- ) mice. Although male and female Mpig6b-/- mice presented with elevated bone marrow megakaryocyte number and macrothrombocytopenia, female Mpig6b-/- mice developed progressive splenomegaly starting at 8 weeks of age. Micro-computed tomography (µCT) of femurs showed that female Mpig6b-/- mice had increased cortical thickness and reduced bone marrow area starting at 8 weeks of age and developed occlusion of the medullary cavity by trabeculae by 16 weeks of age. In contrast, male Mpig6b-/- mice developed only a small number of trabeculae in the medullary cavity at the proximal diaphysis and demonstrated a temporary decrease in bone volume fraction and trabecular thickness at 16 weeks. Ovariectomy of 10-week-old female Mpig6b-/- mice prevented the development of medullary cavity osteosclerosis, whereas orchiectomy of male Mpig6b-/- mice did not exacerbate their disease. Importantly, ovariectomized female Mpig6b-/- mice also demonstrated improvement in spleen weight compared to sham-operated Mpig6b-/- mice, establishing estrogen as a contributing factor to the severity of the megakaryocyte-driven osteosclerosis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Glioma-initiating cells at tumor edge gain signals from tumor core cells to promote their malignancy.

Intratumor spatial heterogeneity facilitates therapeutic resistance in glioblastoma (GBM). Nonetheless, understanding of GBM heterogeneity is largely limited to the surgically resectable tumor core lesion while the seeds for recurrence reside in the unresectable tumor edge. In this study, stratification of GBM to core and edge demonstrates clinically relevant surgical sequelae. We establish regionally derived models of GBM edge and core that retain their spatial identity in a cell autonomous manner. Upon xenotransplantation, edge-derived cells show a higher capacity for infiltrative growth, while core cells demonstrate core lesions with greater therapy resistance. Investigation of intercellular signaling between these two tumor populations uncovers the paracrine crosstalk from tumor core that promotes malignancy and therapy resistance of edge cells. These phenotypic alterations are initiated by HDAC1 in GBM core cells which subsequently affect edge cells by secreting the soluble form of CD109 protein. Our data reveal the role of intracellular communication between regionally different populations of GBM cells in tumor recurrence.

Role of Altered Metabolic Microenvironment in Osteolytic Metastasis.

Metastatic bone disease is generally incurable and leads to pathological fractures, pain, hypercalcemia, spinal cord compression and decreased mobility. The skeleton is the major site of bone metastases from solid cancers, including breast and prostate carcinoma. Bone metastasis is facilitated by activation of bone-resorbing osteoclasts, terminally differentiated multinucleated cells formed by fusion from monocytic precursors. Cancer cells are known to produce specific factors that stimulate osteoclast differentiation and function. Of interest, cancer cells are also known to alter their own bioenergetics increasing the use of glycolysis for their survival and function. Such change in energy utilization by cancer cells would result in altered levels of cell-permeable metabolites, including glucose, lactate, and pyruvate. Osteoclast resorption is energy-expensive, and we have previously demonstrated that during differentiation osteoclasts actively adapt to their bioenergetics microenvironment. We hypothesize that altered bioenergetics state of cancer cells will also modify the bioenergetics substrate availability for the tissue-resident bone cells, potentially creating a favorable milieu for pathological osteolysis. The goals of this review are to analyze how metastasizing cancer cells change the availability of energy substrates in bone microenvironment; and to assess how the altered bioenergetics may affect osteoclast differentiation and activity.

Role of UDP-Sugar Receptor P2Y14 in Murine Osteoblasts.

The purinergic (P2) receptor P2Y14 is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y14 expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca2+]i) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y14 function, we used the P2Y14 inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y14 knockout C2-Ob clones (Y14KO). P2Y14 inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y14 inhibition augmented Ca2+ signalling in response to ATP, ADP and mechanical stimulation. P2Y14 knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPKα phosphorylation. During in vitro osteogenic differentiation, P2Y14 inhibition modulated the timing of osteogenic gene expression, collagen deposition, and mineralization, but did not significantly affect differentiation status by day 28. Of interest, while P2ry14-/- mice from the International Mouse Phenotyping Consortium were similar to wild-type controls in bone mineral density, their tibia length was significantly increased. We conclude that P2Y14 in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation.

Tumor edge-to-core transition promotes malignancy in primary-to-recurrent glioblastoma progression in a PLAGL1/CD109-mediated mechanism.

BACKGROUND: Glioblastoma remains highly lethal due to its inevitable recurrence. Most of this recurrence is found locally, indicating that postsurgical tumor-initiating cells (TICs) accumulate at the tumor edge. These edge-TICs then generate local recurrence harboring new core lesions. Here, we investigated the clinical significance of the edge-to-core (E-to-C) signature generating glioblastoma recurrence and sought to identify its central mediators. METHODS: First, we examined the association of E-to-C-related expression changes to patient outcome in matched primary and recurrent samples (n = 37). Specifically, we tested whether the combined decrease of the edge-TIC marker PROM1 (CD133) with the increase of the core-TIC marker CD109, representing E-to-C transition during the primary-to-recurrence progression, indicates poorer patient outcome. We then investigated the specific molecular mediators that trigger tumor recurrence driven by the E-to-C progression. Subsequently, the functional and translational significance of the identified molecule was validated with our patient-derived edge-TIC models in vitro and in vivo. RESULTS: Patients exhibiting the CD133low/CD109high signature upon recurrence representing E-to-C transition displayed a strong association with poorer progression-free survival and overall survival among all tested patients. Differential gene expression identified that PLAGL1 was tightly correlated with the core TIC marker CD109 and was linked to shorter patient survival. Experimentally, forced PLAGL1 overexpression enhanced, while its knockdown reduced, glioblastoma edge-derived tumor growth in vivo and subsequent mouse survival, suggesting its essential role in the E-to-C-mediated glioblastoma progression. CONCLUSIONS: E-to-C axis represents an ongoing lethal process in primary glioblastoma contributing to its recurrence, partly in a PLAGL1/CD109-mediated mechanism.

Intratumoral spatial heterogeneity of BTK kinomic activity dictates distinct therapeutic response within a single glioblastoma tumor.

OBJECTIVE: Despite significant recent efforts applied toward the development of efficacious therapies for glioblastoma (GBM) through exploration of GBM's genome and transcriptome, curative therapeutic strategies remain highly elusive. As such, novel and effective therapeutics are urgently required. In this study, the authors sought to explore the kinomic landscape of GBM from a previously underutilized approach (i.e., spatial heterogeneity), followed by validation of Bruton's tyrosine kinase (BTK) targeting according to this stepwise kinomic-based novel approach. METHODS: Twelve GBM tumor samples were obtained and characterized histopathologically from 2 patients with GBM. PamStation peptide-array analysis of these tissues was performed to measure the kinomic activity of each sample. The Ivy GBM database was then utilized to determine the intratumoral spatial localization of BTK activity by investigating the expression of BTK-related transcription factors (TFs) within tumors. Genetic inhibition of BTK family members through lentiviral short hairpin RNA (shRNA) knockdown was performed to determine their function in the core-like and edge-like GBM neurosphere models. Finally, the small-molecule inhibitor of BTK, ONO/GS-4059, which is currently under clinical investigation in nonbrain cancers, was applied for pharmacological inhibition of regionally specified newly established GBM edge and core neurosphere models. RESULTS: Kinomic investigation identified two major subclusters of GBM tissues from both patients exhibiting distinct profiles of kinase activity. Comparatively, in these spatially defined subgroups, BTK was the centric kinase differentially expressed. According to the Ivy GBM database, BTK-related TFs were highly expressed in the tumor core, but not in edge counterparts. Short hairpin RNA-mediated gene silencing of BTK in previously established edge- and core-like GBM neurospheres demonstrated increased apoptotic activity with predominance of the sub-G1 phase of core-like neurospheres compared to edge-like neurospheres. Lastly, pharmacological inhibition of BTK by ONO/GS-4059 resulted in growth inhibition of regionally derived GBM core cells and, to a lesser extent, their edge counterparts. CONCLUSIONS: This study identifies significant heterogeneity in kinase activity both within and across distinct GBM tumors. The study findings indicate that BTK activity is elevated in the classically therapy-resistant GBM tumor core. Given these findings, targeting GBM's resistant core through BTK may potentially provide therapeutic benefit for patients with GBM.

Transmission of Mechanical Information by Purinergic Signaling.

The skeleton constantly interacts and adapts to the physical world. We have previously reported that physiologically relevant mechanical forces lead to small repairable membrane injuries in bone-forming osteoblasts, resulting in release of ATP and stimulation of purinergic (P2) calcium responses in neighboring cells. The goal of this study was to develop a theoretical model describing injury-related ATP and ADP release, their extracellular diffusion and degradation, and purinergic responses in neighboring cells. After validation using experimental data for intracellular free calcium elevations, ATP, and vesicular release after mechanical stimulation of a single osteoblast, the model was scaled to a tissue-level injury to investigate how purinergic signaling communicates information about injuries with varying geometries. We found that total ATP released, peak extracellular ATP concentration, and the ADP-mediated signaling component contributed complementary information regarding the mechanical stimulation event. The total amount of ATP released governed spatial factors, such as the maximal distance from the injury at which purinergic responses were stimulated. The peak ATP concentration reflected the severity of an individual cell injury, allowing to discriminate between minor and severe injuries that released similar amounts of ATP because of differences in injury repair, and determined temporal aspects of the response, such as signal propagation velocity. ADP-mediated signaling became relevant only in larger tissue-level injuries, conveying information about the distance to the injury site and its geometry. Thus, we identified specific features of extracellular ATP and ADP spatiotemporal signals that depend on tissue mechanoresilience and encode the severity, scope, and proximity of the mechanical stimulus.

Exosomal Release of L-Plastin by Breast Cancer Cells Facilitates Metastatic Bone Osteolysis.

Bone metastasis from breast and prostate carcinomas is facilitated by activation of bone-resorbing osteoclasts. Using proteomics approaches, we have identified peroxiredoxin-4 (PRDX4) as a cancer-secreted mediator of osteoclastogenesis. We now report characterization of L-plastin in the conditioned media (CM) of MDA-MB-231 human breast cancer cells using immunoblotting and mass spectrometry. The osteoclastogenic potential of MDA-MB-231 CM with siRNA-silenced L-plastin was significantly reduced. L-plastin was detected in cancer-derived exosomes, and inhibition of exosomal release significantly decreased the osteoclastogenic capacity of MDA-MB-231 CM. When added to osteoclast precursors primed with RANKL for 2 days, recombinant L-plastin induced calcium/NFATc1-mediated osteoclastogenesis to the levels similar to continuous treatment with RANKL. Using shRNA, we generated MDA-MB-231 cells lacking L-plastin, PRDX4, or both and injected these cell populations intratibially in CD-1 immunodeficient mice. Micro-CT and histomorphometric analysis demonstrated a complete loss of osteolysis when MDA-MB-231 cells lacking both L-plastin and PRDX4 were injected. A meta-analysis established an increase in L-plastin and PRDX4 mRNA expression in numerous human cancers, including breast and prostate carcinomas. This study demonstrates that secreted L-plastin and PRDX4 mediate osteoclast activation by human breast cancer cells.

A small molecule regulator of tissue transglutaminase conformation inhibits the malignant phenotype of cancer cells.

The protein crosslinking enzyme tissue transglutaminase (tTG) is an acyltransferase which catalyzes transamidation reactions between two proteins, or between a protein and a polyamine. It is frequently overexpressed in several different types of human cancer cells, where it has been shown to contribute to their growth, survival, and invasiveness. tTG is capable of adopting two distinct conformational states: a protein crosslinking active ("open") state, and a GTP-bound, crosslinking inactive ("closed") state. We have previously shown that the ectopic expression of mutant forms of tTG, which constitutively adopt the open conformation, are toxic to cells. This raises the possibility that strategies directed toward causing tTG to maintain an open state could potentially provide a therapeutic benefit for cancers in which tTG is highly expressed. Here, we report the identification of a small molecule, TTGM 5826, which stabilizes the open conformation of tTG. Treatment of breast and brain cancer cell lines, as well as glioma stem cells, with this molecule broadly inhibits their transformed phenotypes. Thus, TTGM 5826 represents the lead compound for a new class of small molecules that promote the toxicity of cancer cells by stabilizing the open state of tTG.

Mechanically stimulated ATP release from murine bone cells is regulated by a balance of injury and repair.

Bone cells sense and actively adapt to physical perturbations to prevent critical damage. ATP release is among the earliest cellular responses to mechanical stimulation. Mechanical stimulation of a single murine osteoblast led to the release of 70 ± 24 amole ATP, which stimulated calcium responses in neighboring cells. Osteoblasts contained ATP-rich vesicles that were released upon mechanical stimulation. Surprisingly, interventions that promoted vesicular release reduced ATP release, while inhibitors of vesicular release potentiated ATP release. Searching for an alternative ATP release route, we found that mechanical stresses induced reversible cell membrane injury in vitro and in vivo. Ca2+/PLC/PKC-dependent vesicular exocytosis facilitated membrane repair, thereby minimizing cell injury and reducing ATP release. Priming cellular repair machinery prior to mechanical stimulation reduced subsequent membrane injury and ATP release, linking cellular mechanosensitivity to prior mechanical exposure. Thus, our findings position ATP release as an integrated readout of membrane injury and repair.