PSMA-targeted radiopharmaceutical therapy is an established treatment option for metastatic castration-resistant prostate cancer (mCRPC). However, response rates and duration using
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Lu-PSMA-617 ...vary considerably between patients. Quantitative
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Lu SPECT imaging is one approach that may be leveraged to more closely monitor inter-cycle response, as well as patient-specific absorbed doses. In this work, we describe our experience implementing quantitative imaging throughout the course of
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Lu-PSMA treatment, including serial SPECT imaging to monitor response and for individualized dosimetry. We also describe our imaging protocols and dose calculation workflows for 3D voxelized patient-specific organ and tumor dosimetry, including a review of the current landscape and efforts towards harmonized dosimetry.
Angiotensin II (Ang II) is a major regulator of steroidogenesis in adrenocortical cells, and is also an effective inducer of cytokine and growth factor synthesis in several cell types. In microarray ...analysis of H295R human adrenocortical cells, the mRNA of brain-derived neurotrophic factor (BDNF), a neurotrophin widely expressed in the nervous system, was one of the most up-regulated genes by Ang II. The aim of the present study was the analysis of the Ang II-induced BDNF expression and BDNF-induced effects in adrenocortical cells. Real-time PCR studies have shown that BDNF is expressed in H295R and rat adrenal glomerulosa cells. In H295R cells, the kinetics of Ang II-induced BDNF expression was faster than that of aldosterone synthase (CYP11B2). Inhibition of calmodulin kinase by KN93 did not significantly affect the Ang II-induced stimulation of BDNF expression, suggesting that it occurs by a different mechanism from the CYP11B2-response. Ang II also caused candesartan-sensitive, type-1 Ang II receptor-mediated stimulation of BDNF gene expression in primary rat glomerulosa cells. In rat adrenal cortex, BDNF protein was localized to the subcapsular region. Ang II increased BDNF protein levels both in human and rat cells, and BDNF secretion of H295R cells. Ang II also increased type-1 Ang II receptor-mediated BDNF expression in vivo in furosemide-treated rats. In rat glomerulosa cells, BDNF induced tropomyosin-related kinase B receptor-mediated stimulation of EGR1 and TrkB expression. These data demonstrate that Ang II stimulates BDNF expression in human and rat adrenocortical cells, and BDNF may have a local regulatory function in adrenal glomerulosa cells.
Angiotensin II stimulates brain-derived neurotrophic factor expression and secretion in adrenal glomerulosa cells, which may activate TrkB receptor-mediated local regulatory mechanisms in this tissue.
Sec14 protein was first identified in Saccharomyces cerevisiae, where it serves as a phosphatidylinositol transfer protein that is essential for the transport of secretory proteins from the Golgi ...complex. A protein domain homologous to Sec14 was identified in several mammalian proteins that regulates Rho GTPases, including exchange factors and GTPase activating proteins. P50RhoGAP, the first identified GTPase activating protein for Rho GTPases, is composed of a Sec14-like domain and a Rho-GTPase activating protein (GAP) domain. The biological function of its Sec14-like domain is still unknown. Here we show that p50RhoGAP is present on endosomal membranes, where it colocalizes with internalized transferrin receptor. We demonstrate that the Sec14-like domain of P50RhoGAP is responsible for the endosomal targeting of the protein. We also show that overexpression of p50RhoGAP or its Sec14-like domain inhibits transferrin uptake. Furthermore, both P50RhoGAP and its Sec14-like domain show colocalization with small GTPases Rab11 and Rab5. We measured bioluminescence resonance energy transfer between p50RhoGAP and Rab11, indicating that these proteins form molecular complex in vivo on endosomal membranes. The interaction was mediated by the Sec 14-like domain of p50RhoGAP. Our results indicate that Sec14-like domain, which was previously considered as a phospholipid binding module, may have a role in the mediation of protein-protein interactions. We suggest that p50RhoGAP provides a link between Rab and Rho GTPases in the regulation of receptor-mediated endocytosis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Dimerization or oligomerization of G-protein-coupled receptors (GPCRs) is a novel concept, which may lead to the reevaluation of the actions of pharmacological ligands, hormones, neurotransmitters, ...and other mediators acting on GPCRs. Although a large number of data obtained using different biophysical, biochemical and structural methods, and functional approaches argue for dimerization or oligomerization of these receptors, several publications criticized the applied methods and challenged the concept. The aim of this paper is to review the data that support the concept of receptor oligomerization, and the most important arguments against it. We conclude that it will require major methodical improvements to obtain decisive proof, whether GPCRs exist in their native membrane environments as homo- or heterodimeric or oligomeric complexes, in which receptor monomers have stable direct interactions. However, overwhelming amounts of data suggest that many GPCRs exhibit functional properties that require direct or indirect interactions between clustered receptors. Although it is difficult to conclude, about the exact nature of these interactions, dimerization or oligomerization of GPCRs is a useful paradigm for pharmacologists to study properties of receptors, which require functionally important clustering of receptors, such as trafficking of newly synthesized receptors to the cell surface, allosteric modulation of ligand binding, signaling specificity, co-internalization, or cross-inhibition of GPCRs.
G protein-coupled receptors are cell surface receptors that mediate the effects of extracellular signals in the endocrine/paracrine and sensory systems. Experimental evidence is accumulating, which ...suggest that these receptors form dimers or higher order oligomers. The functional relevance of G protein-coupled receptor dimerization or oligomerization has been raised in a number of different processes, including ontogeny, internalization, ligand-induced regulation, pharmacological diversity and signal transduction of these receptors. Agonist-independent homo- and hetero-oligomerization of the angiotensin AT1 receptor has been reported, and it has been suggested that hetero-oligomerization with beta-adrenergic receptors leads to cross-inhibition of these receptors. Much less is known about the functional interactions between AT1 receptor homo-oligomers. The aim of the present study was to analyze the functional interactions between these homo-oligomers by determining the functions of normal, AT1 receptor blocker (candesartan) resistant (S109Y) and G protein coupling deficient (DRY/AAY) AT1 receptors (co-)expressed in COS-7 cells. Although we have found no evidence that stimulation of a G protein coupling deficient receptor could cross-activate co-expressed normal receptors, candesartan binding to a signaling deficient receptor caused cross-inhibition of co-expressed candesartan resistant AT1 receptors. Since the studied mutations were in the third intracellular helix of the receptor, the observed effects cannot be explained with domain swapping. These data suggest that AT1 receptor blockers cause cross-inhibition of homo-oligomerized AT1 receptors, and support the concept that receptor dimers/oligomers serve as the functional unit of G protein-coupled receptors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
2078 Background: Immune checkpoint inhibition with pembrolizumab has shown clinical benefits in multiple cancer types. However, success with checkpoint inhibition remains elusive in glioblastoma ...(GBM). Identifying patients a priori who would benefit from checkpoint inhibition with pembrolizumab in GBM may allow for improved survival. Currently there are no immune biomarkers that allow for selective treatment with pembrolizumab in GBM. Methods: The exploratory endpoint for our prospective single-arm study (NCT 03347617) evaluated longitudinal changes in immune cell sub-populations (B-cells, T-cells, NK cells, monocytes, and myeloid cells) in 50 newly diagnosed GBM patients who received standard of care chemoradiation with concurrent pembrolizumab after maximal safe resection. Flow cytometry was performed on blood collected at predetermined timepoints (pre-treatment, post-radiation, suspected progression, and confirmed progression) and immune cell sub-populations were evaluated as covariates in univariate and multivariate overall survival analysis while stratifying by IDH and MGMT methylation status and controlling for age and KPS using Cox proportional hazards regression in R. Results: Of the 56 patients enrolled in this trial, 50 were included in the analysis who had completed the pre-treatment blood collection. Median overall survival was 13.84 months. Nineteen patients (38%) were female and 31 (62%) were male. Median survival was 58 years (IQR: 46.25-69). Median KPS at study start was 90 (IQR: 80-90). MGMT methylation status included 23 (46%) patients not methylation and 25 (50%) hypermethylated patients, as well as 2 (4%) patients unidentified. IDH status included 39 (78%) wild-type patients, 10 (20%) mutant patients and 1 (2%) unidentified. At the pre-treatment timepoint, univariate analysis identified the following serum derived immune populations as being significantly associated with overall survival: cytotoxic T-cells (CD3+CD8+; HR: 0.98; p=0.094); CD4:CD8 ratio (HR:1.35; p=0.003); regulatory T-cells (4+127-BRIGHT25+; HR: 0.99; p=0.075); B-cells% (CD19+; HR:1.07; p=0.045). Multivariate analysis revealed significant association of CD4:CD8 ratio (HR:1.80; p=0.002) and regulatory T-cells (HR:0.98; p=0.015) with overall survival. Conclusions: GBM patients with increased serum CD4:CD8 ratios are associated with worsened overall survival prior to receiving standard of care chemoradiation and pembrolizumab. These results suggest that pembrolizumab should be withheld in patients with pre-treatment elevated levels of CD4:CD8 ratios. Alternatively, patients with higher serum regulatory T-cells have improved overall survival and may benefit from the addition of pembrolizumab. Validation of these results is needed in patients treated with standard of care versus standard of care plus pembrolizumab before implementing into clinical practice. Clinical trial information: NCT03347617 .
Abstract
BACKGROUND
Standard of care (SOC) for newly diagnosed glioblastoma includes radiotherapy plus temozolomide followed by 6 adjuvant cycles monthly temozolomide per the Stupp protocol. We ...report the secondary objectives of a single arm phase 2 study evaluating the clinical impact of the addition of immune check point inhibitor pembrolizumab to this standard therapy.
METHODS
Patients with histopathology proven glioblastoma (WHO 4) eligible to receive SOC were prospectively enrolled to received pembrolizumab 200 mg iv every 3 weeks for up to 35 doses, progression, development of dose limiting toxicity or withdrawal of consent. Patients also receive ferumoxytol steady state MRI scans at baseline, 4 weeks after the last day of standard of care stereotactic radiosurgery or chemoradiotherapy, every 9 weeks thereafter until suspected radiographic progression, and then within 4 weeks from suspected radiographic progression. Primary endpoints of the study were to test the sensitivity and specificity of ferumoxytol steady state imaging for response assessment in this cohort. Secondary objective includes Progression free (PFS) and Overall survival (OS) and toxicity. PFS and OS were analyzed using Kaplan-Meier method.
RESULTS
A total of 52 patients (19 F, 33 M; 43 IHD wt and 9 IDH mu; 25 MGMT gene methylated, 27 unmethylated) were enrolled and included. At a median Follow up of 14.3 months, median PFS was 10.7 m (95% CI:8.1-12.6) and median OS was 14.4 m (95%CI: 12.5-16.9). Two grade 3 or higher toxicities (bullous dermatitis and Steven-Johnson syndrome) were attributed to pembrolizumab.
CONCLUSIONS
Although, the addition of pembrolizumab to SOC was well tolerated in most patients, this study did not see any meaning clinical benefit in PFS or OS with the addition of pembrolizumab in patients with newly diagnosed glioblastoma. Analysis for the primary imaging endpoint is ongoing.
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