HPK1 citron homology domain regulates phosphorylation of SLP76 and modulates kinase domain interaction dynamics.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell receptor signaling and as such is an attractive target for cancer immunotherapy. Although the role of the HPK1 kinase domain (KD) has been extensively characterized, the function of its citron homology domain (CHD) remains elusive. Through a combination of structural, biochemical, and mechanistic studies, we characterize the structure-function of CHD in relationship to KD. Crystallography and hydrogen-deuterium exchange mass spectrometry reveal that CHD adopts a seven-bladed ß-propellor fold that binds to KD. Mutagenesis associated with binding and functional studies show a direct correlation between domain-domain interaction and negative regulation of kinase activity. We further demonstrate that the CHD provides stability to HPK1 protein in cells as well as contributes to the docking of its substrate SLP76. Altogether, this study highlights the importance of the CHD in the direct and indirect regulation of HPK1 function.

Dyskinesia-hyperpyrexia syndrome in Parkinson's disease triggered by overdose of levodopa - a case report and literature review.

Dyskinesia-hyperpyrexia syndrome, a rare medical emergency in Parkinson's disease, is first described in 2010. It is characterized by severe continuous dyskinesia associated with rhabdomyolysis, hyperthermia and subsequent alteration of the mental state. Gradual reduction of dopaminergic dose or DBS is recommended treatment. The prognosis is usually good, but sometimes fatal. But so far, this potentially fatal complication is not widely recognized by clinicians. In emergency, if clinicians fail to make prompt diagnosis and treatment, patients' conditions may get worse, and their lives may be threatened in serious cases.

Filtered air intervention modulates hypothalamic-pituitary-thyroid/gonadal axes by attenuating inflammatory responses in adult rats after fine particulate matter (PM2.5) exposure.

We have previously reported that filtered air (FA) intervention reduces inflammation and hypothalamus-pituitary-adrenal axis activation after fine particulate matter (PM2.5 exposure). Whether FA also modulates the hypothalamic-pituitary-thyroid (HPT) and hypothalamic-pituitary-gonadal (HPG) axes in rats after PM2.5 exposure is still unknown. Adult Sprague-Dawley rats were exposed to PM2.5 by using a "real-world" PM2.5 exposure system, and the FA intervention was conducted by renewing for 15 days. PM2.5 inhalation decreased thyrotropin-releasing hormone (TRH) and thyroxine (T4) levels in both male and female rats, and thyroid-stimulating hormone (TSH) level in male rats. FA intervention attenuated the reduction in TRH and TSH levels in male rats and reduction in T4 level in female rats. PM2.5 inhalation also reduced testosterone (T) level in male rats, and estradiol (E2) and progesterone (PROG) levels in female rats, and these changes were attenuated after FA intervention. The FA intervention attenuated the decreases in CD8 T cells and T cells induced by PM2.5 inhalation in female rats only by flow cytometry analysis. In blood, FA interventions ameliorated IL-6 and IL-1ß mRNA levels in both male and female rats after PM2.5 exposure. FA intervention restored the IL-4 and IL-10 levels in female rats after PM2.5 exposure. Moreover, FA intervention ameliorated the inflammatory responses induced by PM2.5 inhalation in the thyroid and gonads in both male and female rats. These data indicate that FA intervention exerted an effect on modulating the hormonal balance of the HPT and HPG axes, and this may be related to a reduction in the inflammatory responses in the thyroid and gonads of PM2.5-treated rats, respectively.

Hypoxia-Inducible Factor 1α and 2α Have Beneficial Effects in Remote Ischemic Preconditioning Against Stroke by Modulating Inflammatory Responses in Aged Rats.

Limb remote ischemic preconditioning (RIPC) has been proven to alleviate stroke injury in young rats, but its protective effect and its mechanism in aged rats are still unclear. Hypoxia-inducible factor (HIF) is one of the important markers of stroke, and its high expression plays an important role in the pathogenesis of stroke. In this study, we tested the hypothesis that RIPC could regulate the expression of HIF, leading to reduced inflammatory responses in aged rats. Stroke was induced by transient middle cerebral artery occlusion (MCAo) in aged rats, and RIPC was conducted in both hind limbs. The HIF-1α and HIF-2α mRNA and protein were examined by real-time RT-PCR and western blotting (WB). Inflammatory cytokines in the peripheral blood and brain were measured using AimPlex multiplex immunoassays. The protein levels of p-Akt, Akt, p-ERK, and ERK were examined by WB. We investigated that RIPC reduced the infarct size, improved neurological functions, and decreased the expression of HIF-1α and HIF-2α in the ischemic brain. RIPC reduced the levels of IL-1ß, IL-6 and IFN-γ in the peripheral blood and the levels of IL-1ß and IFN-γ in the ischemic brain 48 h post-stroke. Moreover, intraperitoneal injection of the HIF inhibitor, acriflavine hydrochloride (ACF), abolished the protection of RIPC with respect to infarct size and neurological functions and neutralized the downregulation of pro-inflammatory IL-1ß, IL-6 and IFN-γ. ACF also reversed the activation of the Akt signaling pathway induced by RIPC following stroke. HIF may play a key role in RIPC, which was likely mediated by the Akt signaling pathway and systemic modulation of the inflammatory response in aged rats.

CD96 functions as a co-stimulatory receptor to enhance CD8+ T cell activation and effector responses.

CD96 is a member of the poliovirus receptor (PVR, CD155)-nectin family that includes T cell Ig and ITIM domain (TIGIT) and CD226. While CD96, TIGIT, and CD226 have important roles in regulating NK cell activity, and TIGIT and CD226 have also been shown to regulate T cell responses, it is unclear whether CD96 has inhibitory or stimulatory function in CD8+ T cells. Here, we demonstrate that CD96 has co-stimulatory function on CD8+ T cells. Crosslinking of CD96 on human or mouse CD8+ T cells induced activation, effector cytokine production, and proliferation. CD96 was found to transduce its activating signal through the MEK-ERK pathway. CD96-mediated signaling led to increased frequencies of NUR77- and T-bet-expressing CD8+ T cells and enhanced cytotoxic effector activity, indicating that CD96 can modulate effector T cell differentiation. Antibody blockade of CD96 or genetic ablation of CD96 expression on CD8+ T cells impaired expression of transcription factors and proinflammatory cytokines associated with CD8+ T cell activation in in vivo models. Taken together, CD96 has a co-stimulatory role in CD8+ T cell activation and effector function.

Peripheral T cell expansion predicts tumour infiltration and clinical response.

Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL11, the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response.

MiR-22-3p Regulates Amyloid ß Deposit in Mice Model of Alzheimer's Disease by Targeting Mitogen-activated Protein Kinase 14.

Propose: To investigate whether miR-22-3p is able to regulate AD development and its molecular mechanism. METHODS: Morris water maze test was performed to test the spatial memory. Quantitative polymerase chain reaction (qPCR) was used to assess the expression level of miR-22-3p. The enzymelinked immunosorbent assay (ELISA) was used to assess the levels of Aß40 and Aß42. Immunoblotting analysis was performed to detect the protein expression levels of amyloid precursor protein (APP), mitogen-activated protein kinase 14 (MAPK14) and beta-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1). Luciferase assay was used to identify the interaction between miR- 22-3p and MAPK14. The tetrazolium dye (MTT) colorimetric assay was used to test the influence of miR-22-3p overexpression on cell viability. Flow cytometry analysis was performed to evaluate the effect of miR-22-3p overexpression on cell apoptosis. RESULTS: Morris water maze test showed that mice model of AD had impaired spatial memory, which was able to be ameliorated by miR-22-3p overexpression. Immunoblotting analysis revealed that the protein expression levels of APP, MAPK14 and BACE1 were enhanced in AD model, which could be prevented by miR-22-3p overexpression. ELISA showed that Aß40 and Aß42 levels were dramatically increased in AD model, which were inhibited by miR-22-3p overexpression. Luciferase assay and immunoblotting analysis indicated that miR-22-3p targeted and regulated MAPK14 expression. CONCLUSION: MiR-22-3p overexpression reduced Aß deposit and alleviated AD symptoms by targeting and regulating MAPK14 expression, which ameliorated AD symptoms.

CD226 regulates natural killer cell antitumor responses via phosphorylation-mediated inactivation of transcription factor FOXO1.

Natural killer (NK) cell recognition of tumor cells is mediated through activating receptors such as CD226, with suppression of effector functions often controlled by negative regulatory transcription factors such as FOXO1. Here we show that CD226 regulation of NK cell cytotoxicity is facilitated through inactivation of FOXO1. Gene-expression analysis of NK cells isolated from syngeneic tumors grown in wild-type or CD226-deficient mice revealed dysregulated expression of FOXO1-regulated genes in the absence of CD226. In vitro cytotoxicity and stimulation assays demonstrated that CD226 is required for optimal killing of tumor target cells, with engagement of its ligand CD155 resulting in phosphorylation of FOXO1. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Furthermore, inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses. These results define a pathway by which CD226 exerts control of NK cell responses against tumors.

The Kinase Activity of Hematopoietic Progenitor Kinase 1 Is Essential for the Regulation of T Cell Function.

We examined hematopoietic protein kinase 1 (HPK1), whose reliance on scaffold versus kinase functions for negative immune cell regulation is poorly understood and critical to its assessment as a viable drug target. We identify kinase-dependent roles for HPK1 in CD8 T cells that restrict their anti-viral and anti-tumor responses by using HPK1 kinase-dead (HPK1.kd) knockin mice. Loss of HPK1 kinase function enhanced T cell receptor signaling and cytokine secretion in a T-cell-intrinsic manner. In response to chronic lymphocytic choriomeningitis virus (LCMV) infection or tumor challenge, viral clearance and tumor growth inhibition were enhanced in HPK1.kd mice, accompanied by an increase in effector CD8 T cell function. Co-blockade of PD-L1 further enhanced T effector cell function, resulting in superior anti-viral and anti-tumor immunity over single target blockade. These results identify the importance of HPK1 kinase activity in the negative regulation of CD8 effector functions, implicating its potential as a cancer immunotherapy target.

Hypoxia Inducible Factor 1α Plays a Key Role in Remote Ischemic Preconditioning Against Stroke by Modulating Inflammatory Responses in Rats.

BACKGROUND: Limb remote ischemic preconditioning (RIPC) protects against brain injury induced by stroke, but the underlying protective mechanisms remain unknown. As hypoxia inducible factor 1α (HIF-1α) is neuroprotective in stroke and mediates neuroinflammation, we tested the hypothesis that HIF-1α is a key factor of RIPC against stroke by mediating inflammation. METHODS AND RESULTS: Stroke was induced by transient middle cerebral artery occlusion in rats, and RIPC was conducted in both hind limbs. The HIF-1α mRNA was examined by quantitative reverse transcription polymerase chain reaction after RIPC. In addition, inflammatory cytokines in the peripheral blood and brain were measured using the AimPlex multiplex immunoassays. Data showed that RIPC reduced the infarct size, improved neurological functions, and increased HIF-1α mRNA levels, interleukin (IL)-4, and IL-10 protein levels in the peripheral blood. Intraperitoneal injection of the HIF activator, dimethyloxaloylglycine, reduced the infarct size and inhibited interferon-γ protein levels, while promoting IL-4 and IL-10 protein levels, while decreasing interferon-γ protein levels in both the peripheral blood and ischemic brain. In addition, injection of dimethyloxaloylglycine had a synergistic effect with RIPC on reducing infarction and improving neurological functions, as well as decreasing interferon-γ in the peripheral blood and ischemic brain. In contrast, injection of the HIF inhibitor, acriflavine hydrochloride, abolished the protective effects of RIPC on infarction, and reduced IL-4 and IL-10 protein levels in both the peripheral blood and ischemic brain. CONCLUSIONS: We conclude that HIF-1α plays a key role in RIPC, likely mediated by a systemic modulation of the inflammatory response.

The cerebral circulation and cerebrovascular disease III: Stroke.

In this paper, our review series on cerebrovascular disease anatomy, physiology, and pathology ends with a thorough discussion of the most significant cerebrovascular pathology: stroke. This discussion proceeds through two layers of organization. First, stroke is divided up into its main etiologic categories (ischemic stroke/transient ischemic attack, hemorrhagic stroke, and ischemic to hemorrhagic transformation). Then, the epidemiological, pathophysiological, clinical, and therapeutic (employed currently as well as emerging) aspects of each etiology are explored; emphasis is placed upon the therapeutic aspects. Finally, once we have covered all aspects of each etiologic category, we end our review with a defense of the thesis that there is much hope for the future of stroke treatment to be derived from familiarity with the literature on emerging therapies.

Metabolite profiling stratifies pancreatic ductal adenocarcinomas into subtypes with distinct sensitivities to metabolic inhibitors.

Although targeting cancer metabolism is a promising therapeutic strategy, clinical success will depend on an accurate diagnostic identification of tumor subtypes with specific metabolic requirements. Through broad metabolite profiling, we successfully identified three highly distinct metabolic subtypes in pancreatic ductal adenocarcinoma (PDAC). One subtype was defined by reduced proliferative capacity, whereas the other two subtypes (glycolytic and lipogenic) showed distinct metabolite levels associated with glycolysis, lipogenesis, and redox pathways, confirmed at the transcriptional level. The glycolytic and lipogenic subtypes showed striking differences in glucose and glutamine utilization, as well as mitochondrial function, and corresponded to differences in cell sensitivity to inhibitors of glycolysis, glutamine metabolism, lipid synthesis, and redox balance. In PDAC clinical samples, the lipogenic subtype associated with the epithelial (classical) subtype, whereas the glycolytic subtype strongly associated with the mesenchymal (QM-PDA) subtype, suggesting functional relevance in disease progression. Pharmacogenomic screening of an additional ∼ 200 non-PDAC cell lines validated the association between mesenchymal status and metabolic drug response in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity of tumors to a variety of metabolic inhibitors.

The Selective PI3K Inhibitor XL147 (SAR245408) Inhibits Tumor Growth and Survival and Potentiates the Activity of Chemotherapeutic Agents in Preclinical Tumor Models.

Dysregulation of PI3K/PTEN pathway components, resulting in hyperactivated PI3K signaling, is frequently observed in various cancers and correlates with tumor growth and survival. Resistance to a variety of anticancer therapies, including receptor tyrosine kinase (RTK) inhibitors and chemotherapeutic agents, has been attributed to the absence or attenuation of downregulating signals along the PI3K/PTEN pathway. Thus, PI3K inhibitors have therapeutic potential as single agents and in combination with other therapies for a variety of cancer indications. XL147 (SAR245408) is a potent and highly selective inhibitor of class I PI3Ks (α, ß, γ, and δ). Moreover, broad kinase selectivity profiling of >130 protein kinases revealed that XL147 is highly selective for class I PI3Ks over other kinases. In cellular assays, XL147 inhibits the formation of PIP3 in the membrane, and inhibits phosphorylation of AKT, p70S6K, and S6 in multiple tumor cell lines with diverse genetic alterations affecting the PI3K pathway. In a panel of tumor cell lines, XL147 inhibits proliferation with a wide range of potencies, with evidence of an impact of genotype on sensitivity. In mouse xenograft models, oral administration of XL147 results in dose-dependent inhibition of phosphorylation of AKT, p70S6K, and S6 with a duration of action of at least 24 hours. Repeat-dose administration of XL147 results in significant tumor growth inhibition in multiple human xenograft models in nude mice. Administration of XL147 in combination with chemotherapeutic agents results in antitumor activity in xenograft models that is enhanced over that observed with the corresponding single agents.

Cognitive impairments associated with corpus callosum infarction: a ten cases study.

The aim of this study was to determine whether the cognitive impairment is associated with corpus callosum infarctions. Ten corpus callosum infarction patients were enrolled in this study. Their emotions, cognitive and language abilities, memory, comprehensive perception were assessed using the Chinese version of following measures: Mini Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), World Health Organization-University of California-Los Angeles Auditory Verbal Learning Test (WHO-UCLA AVLT), Wechsler Adult Intelligence Scale (WAIS) Digit Span subtest and so on. The same measurements were performed on healthy control participants as contrast for analysis. Infarction most frequently occurred in the body and/or splenium of the corpus callosum. The scores of the most cognitive tests in the corpus callosum infarction patients were significantly worse than those of the control participants (P<0.05). Except for the naming ability, the patients showed significantly poorer performance at the overall level of MMSE than the controls did (P<0.05). Consistently, the results of MoCA suggested a significant reduction in visuospatial abilities of execution, orientation, attention, calculation, delayed memory, language, and repetition capabilities in the patients with respect to the control (P<0.05). In addition, the scores in the case group were significantly worse than those in the control group in the auditory word learning test, digital span and Rey complex figure test (P<0.05). Corpus callosum infarction can cause cognitive dysfunction, which poses obstacles to memory in the acute phase, accompanied by different degrees of decline in visuospatial abilities, attention and calculating abilities.

MMP-1 and Pro-MMP-10 as potential urinary pharmacodynamic biomarkers of FGFR3-targeted therapy in patients with bladder cancer.

PURPOSE: The aim of this study was to identify noninvasive pharmacodynamic biomarkers of FGFR3-targeted therapies in bladder cancer to facilitate the clinical development of experimental agent targeting FGFR3. EXPERIMENTAL DESIGN: Potential soluble pharmacodynamic biomarkers of FGFR3 were identified using a combination of transcriptional profiling and biochemical analyses in preclinical models. Two matrix metalloproteinases (MMP), MMP-1 and MMP-10, were selected for further studies in human bladder cancer xenograft models treated with a specific anti-FGFR3 monoclonal antibody, R3Mab. Serum and urinary levels of MMP-1 and MMP-10 were determined in healthy donors and patients with bladder cancer. The modulation of MMP-1 and MMP-10 by R3Mab in patients with bladder cancer was further evaluated in a phase I dose-escalation study. RESULTS: MMP-1 and MMP-10 mRNA and protein were downmodulated by FGFR3 shRNA and R3Mab in bladder cancer cell lines. FGFR3 signaling promoted the expression and secretion of MMP-1 and pro-MMP-10 in a MEK-dependent fashion. In bladder cancer xenograft models, R3Mab substantially blocked tumor progression and reduced the protein levels of human MMP-1 and pro-MMP-10 in tumor tissues as well as in mouse serum. Furthermore, both MMP-1 and pro-MMP-10 were elevated in the urine of patients with advanced bladder cancer. In a phase I dose-escalation trial, R3Mab administration resulted in an acute reduction of urinary MMP-1 and pro-MMP-10 levels in patients with bladder cancer. CONCLUSION: These findings reveal a critical role of FGFR3 in regulating MMP-1 and pro-MMP-10 expression and secretion, and identify urinary MMP-1 and pro-MMP-10 as potential pharmacodynamic biomarkers for R3Mab in patients with bladder cancer.

Characterization of the activity of the PI3K/mTOR inhibitor XL765 (SAR245409) in tumor models with diverse genetic alterations affecting the PI3K pathway.

Activation of the PI3K (phosphoinositide 3-kinase) pathway is a frequent occurrence in human tumors and is thought to promote growth, survival, and resistance to diverse therapies. Here, we report pharmacologic characterization of the pyridopyrimidinone derivative XL765 (SAR245409), a potent and highly selective pan inhibitor of class I PI3Ks (α, ß, γ, and δ) with activity against mTOR. Broad kinase selectivity profiling of >130 protein kinases revealed that XL765 is highly selective for class I PI3Ks and mTOR over other kinases. In cellular assays, XL765 inhibits the formation of PIP(3) in the membrane, and inhibits phosphorylation of AKT, p70S6K, and S6 phosphorylation in multiple tumor cell lines with different genetic alterations affecting the PI3K pathway. In a panel of tumor cell lines, XL765 inhibits proliferation with a wide range of potencies, with evidence of an impact of genotype on sensitivity. In mouse xenograft models, oral administration of XL765 results in dose-dependent inhibition of phosphorylation of AKT, p70S6K, and S6 with a duration of action of approximately 24 hours. Repeat dose administration of XL765 results in significant tumor growth inhibition in multiple human xenograft models in nude mice that is associated with antiproliferative, antiangiogenic, and proapoptotic effects.

FGFR3 stimulates stearoyl CoA desaturase 1 activity to promote bladder tumor growth.

Fibroblast growth factor receptor 3 (FGFR3) belongs to a family of receptor tyrosine kinases that control cell proliferation, differentiation, and survival. Aberrant activation of FGFR3 via overexpression or mutation is a frequent feature of bladder cancer; however, its molecular and cellular consequences and functional relevance to carcinogenesis are not well understood. Through transcriptional profiling of bladder carcinoma cells subjected to short hairpin RNA knockdown of FGFR3, we identified a gene-signature linking FGFR3 signaling with de novo sterol and lipid biosynthesis and metabolism. We found that FGFR3 signaling promotes the cleavage and activation of the master transcriptional regulator of lipogenesis, sterol regulatory element-binding protein 1(SREBP1/SREBF1), in a PI3K-mTORC1-dependent fashion. In turn, SREBP1 regulates the expression of key lipogenic enzymes, including stearoyl CoA desaturase 1 (SCD1/SCD). SCD1 is the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids and is crucial for lipid homeostasis. In human bladder cancer cell lines expressing constitutively active FGFR3, knockdown of SCD1 by siRNA markedly attenuated cell-cycle progression, reduced proliferation, and induced apoptosis. Furthermore, inducible knockdown of SCD1 in a bladder cancer xenograft model substantially inhibited tumor progression. Pharmacologic inhibition of SCD1 blocked fatty acid desaturation and also exerted antitumor activity in vitro and in vivo. Together, these findings reveal a previously unrecognized role of FGFR3 in regulating lipid metabolism to maintain tumor growth and survival, and also identify SCD1 as a potential therapeutic target for FGFR3-driven bladder cancer.

Antibody-based targeting of FGFR3 in bladder carcinoma and t(4;14)-positive multiple myeloma in mice.

Overexpression of FGF receptor 3 (FGFR3) is implicated in the development of t(4;14)-positive multiple myeloma. While FGFR3 is frequently overexpressed and/or activated through mutations in bladder cancer, the functional importance of FGFR3 and its potential as a specific therapeutic target in this disease have not been elucidated in vivo. Here we report that inducible knockdown of FGFR3 in human bladder carcinoma cells arrested cell-cycle progression in culture and markedly attenuated tumor progression in xenografted mice. Further, we developed a unique antibody (R3Mab) that inhibited not only WT FGFR3, but also various mutants of the receptor, including disulfide-linked cysteine mutants. Biochemical analysis and 2.1-A resolution crystallography revealed that R3Mab bound to a specific FGFR3 epitope that simultaneously blocked ligand binding, prevented receptor dimerization, and induced substantial conformational changes in the receptor. R3Mab exerted potent antitumor activity against bladder carcinoma and t(4;14)-positive multiple myeloma xenografts in mice by antagonizing FGFR3 signaling and eliciting antibody-dependent cell-mediated cytotoxicity (ADCC). These studies provide in vivo evidence demonstrating an oncogenic role of FGFR3 in bladder cancer and support antibody-based targeting of FGFR3 in hematologic and epithelial cancers driven by WT or mutant FGFR3.