We report the first small-molecule protease-activated receptor (PAR) 2 agonists, AC-55541 N-1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl-benzamide ...and AC-264613 2-oxo-4-phenylpyrrolidine-3-carboxylic acid 1-(3-bromo-phenyl)-(E/Z)-ethylidene-hydrazide, each representing a distinct chemical series. AC-55541 and AC-264613 each activated PAR2 signaling in cellular proliferation assays, phosphatidylinositol hydrolysis assays, and Ca(2+) mobilization assays, with potencies ranging from 200 to 1000 nM for AC-55541 and 30 to 100 nM for AC-264613. In comparison, the PAR2-activating peptide 2-furoyl-LIGRLO-NH(2) had similar potency, whereas SLIGRL-NH(2) was 30 to 300 times less potent. Neither AC-55541 nor AC-264613 had activity at any of the other PAR receptor subtypes, nor did they have any significant affinity for over 30 other molecular targets involved in nociception. Visualization of EYFP-tagged PAR2 receptors showed that each compound stimulated internalization of PAR2 receptors. AC-55541 and AC-264613 were well absorbed when administered intraperitoneally to rats, each reaching micromolar peak plasma concentrations. AC-55541 and AC-264613 were each stable to metabolism by liver microsomes and maintained sustained exposure in rats, with elimination half-lives of 6.1 and 2.5 h, respectively. Intrapaw administration of AC-55541 or AC-264613 elicited robust and persistent thermal hyperalgesia and edema. Coadministration of either a tachykinin 1 (neurokinin 1) receptor antagonist or a transient receptor potential vanilloid (TRPV) 1 antagonist completely blocked these effects. Systemic administration of either AC-55541 or AC-264613 produced a similar degree of hyperalgesia as was observed when the compounds were administered locally. These compounds represent novel small-molecule PAR2 agonists that will be useful in probing the physiological functions of PAR2 receptors.
We expressed the cloned mu-opioid receptor (muR) in high abundance (5.5 x 10(6) sites/cell) with an amino-terminal epitope tag (EYMPME) in human embryonic kidney 293 cells. The epitope-tagged ...receptor (EE-muR) was similar to the untagged mu R ligand binding and agonist-dependent inhibition of cyclic AMP accumulation. By confocal microscopy, the labeled receptor was shown to be largely confined to the plasma membrane. Pretreatment with morphine failed to affect the cellular distribution of the receptor as judged by immunofluorescence and tracer binding studies. In contrast, exposure to the mu-specific peptide agonist D-Ala2, MePhe4, Gly-ol5enkephalin (DAMGO) caused strong labeling of endocytic vesicles, indicating extensive agonist-induced cellular redistribution of EE-muR. Tracer binding studies suggested partial net internalization and a small degree of down-regulation caused by DAMGO. EE-muR-containing membranes were solubilized in detergent 3-(3-cholamidopropyl) dimethylammonio-1-propanesulfonate and immunoprecipitated by an anti-epitope monoclonal antibody. Immunoblotting revealed a prominent band at approximately 70 kDa with weaker bands at approximately 65 kDa. EE-muR was labeled with gamma-32PATP in permeabilized cells, immunoprecipitated, and analyzed by polyacrylamide gel electrophoresis autoradiography. A prominent band at 65-70 kDa indicated the presence of basal receptor phosphorylation occurring in the absence of agonist, which was enhanced approximately 1.8-fold with the addition of morphine. In conclusion, intracellular trafficking of the muR appears to depend on the agonist, with morphine and DAMGO having markedly different effects. Unlike other G protein-coupled receptors, basal phosphorylation is substantial, even in the absence of agonist.
PD-1/L1 axis-directed therapies produce clinical responses in a subset of patients; therefore, biomarkers of response are needed. We hypothesized that quantifying key immunosuppression mechanisms ...within the tumor microenvironment by multiparameter algorithms would identify strong predictors of anti-PD-1 response.
Pretreatment tumor biopsies from 166 patients treated with anti-PD-1 across 10 academic cancer centers were fluorescently stained with multiple markers in discovery (
= 24) and validation (
= 142) cohorts. Biomarker-positive cells and their colocalization were spatially profiled in pathologist-selected tumor regions using novel Automated Quantitative Analysis algorithms. Selected biomarker signatures, PD-1/PD-L1 interaction score, and IDO-1/HLA-DR coexpression were evaluated for anti-PD-1 treatment outcomes.
In the discovery cohort, PD-1/PD-L1 interaction score and/or IDO-1/HLA-DR coexpression was strongly associated with anti-PD-1 response (
= 0.0005). In contrast, individual biomarkers (PD-1, PD-L1, IDO-1, HLA-DR) were not associated with response or survival. This finding was replicated in an independent validation cohort: patients with high PD-1/PD-L1 and/or IDO-1/HLA-DR were more likely to respond (
= 0.0096). These patients also experienced significantly improved progression-free survival (HR = 0.36;
= 0.0004) and overall survival (HR = 0.39;
= 0.0011). In the combined cohort, 80% of patients exhibiting higher levels of PD-1/PD-L1 interaction scores and IDO-1/HLA-DR responded to PD-1 blockers (
= 0.000004). In contrast, PD-L1 expression was not predictive of survival.
Quantitative spatial profiling of key tumor-immune suppression pathways by novel digital pathology algorithms could help more reliably select melanoma patients for PD-1 monotherapy.
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Background Q-F (NCT02668653) showed that the highly potent, selective, type 2 FLT3 inhibitor quizartinib (Q) + standard chemotherapy ± transplantation, followed by Q monotherapy for ≥36 cycles, ...reduced the relative risk of death by 22.4% vs placebo (P) in newly diagnosed (nd) FLT3-ITD+ AML, with HR of 0.776 and P value of 0.0324 (PMID: 37116523). In Q-F, FLT3-ITD mutation status was determined using a FLT3-ITD mutation detection clinical trial assay (CTA) validated under design control by Navigate BioPharma Services, Inc. We describe the results of the bridging study, aimed to show agreement between the CTA & the LeukoStrat CDx FLT3 Mutation Assay (CDx, by Invivoscribe) in FLT3-ITD+ pt selection and to determine if Q efficacy (overall survival OS) was maintained in nd FLT3-ITD+ AML pts from Q-F, if pts had been selected using the CDx. Methods In both CTA & CDx, DNA, extracted from bone marrow (n=884 each) or peripheral blood (n=139 each), was amplified via PCR and amplicons were detected via capillary electrophoresis. A sample was considered CTA+ if the variant allele frequency ( FLT3-ITD/total FLT3) was ≥3% and CDx+ if the signal ratio (SR; FLT3-ITD/ FLT3 WT) was ≥0.05. The agreement between CTA & CDx was based on evaluating CTA+ & CTA− samples with the CDx assay. A primary analysis included the CDx detected (CDx+ & CDx−) and the CDx invalid results. A secondary analysis used CDx+ and CDx− results only. To establish agreement between CDx & CTA, positive % agreement (PPA) and negative % agreement (NPA) were determined using CTA results as reference for the agreement analysis set (AAS). Concordance was established if the lower bounds of the 95% CIs for both PPA & NPA exceeded 90% for the analysis that included the invalid CDx results. Median OS in the subgroups was calculated based on Kaplan-Meier estimates. Stratified Cox proportional hazards regression model was used to estimate HRs, 95% CI, and P value. Results Full analysis set (N=3468) included all Q-F screened CTA+ pts (n=863), all screened CTA− pts (n=2556), and pts with unknown CTA status not eligible for randomization due to other criteria (n=49). Of these, 1032 pts formed the primary analysis set (PAS), including all pts randomized in Q-F with samples available for CDx testing (N=513: Q, n=254; P, n=259) and a randomly selected subset of CTA− pts (n=519). The ascertainment rate was 95.2% (513/539), as 26 of the 539 pts randomized in Q-F were excluded from the bridging study. Within the PAS, 3 samples were not tested by CDx due to insufficient volume/DNA amount. The AAS (N=1029: CTA+, n=513; CTA−, n=516) included pts in the PAS with valid CTA results and tested with CDx. In the AAS, 6 samples (3 CTA+, 3 CTA−) did not yield valid CDx results, resulting in 1023 CDx-evaluable total pts (CTA+, n=510; CTA−, n=513). Among 510 CTA+ samples, 483 were CDx+. Among 513 CTA− samples, 513 were CDx−. Therefore, 996 samples yielded concordant results, 27 samples yielded discordant results, and 6 samples did not yield a valid CDx result for comparison. Point estimates of PPA & NPA were 94.2% & 99.4%, respectively, with invalid CDx results included in the calculation, and 94.7% & 100%, respectively, without invalid CDx results. The lower bounds of the 95% CIs were all above the corresponding acceptance criterion of 90% for PPA & NPA (Table 1). In Q-F, the prevalence of CTA+ was 24.9% among screened pts (863/3468), whereas in the bridging study, 49.9% (510/1023) of pts were CTA+: this enrichment in CTA+ pts could lead to a biased estimate of the agreement between CDx & CTA when using CDx as reference. The positive predictive value (PPV) and negative predictive value (NPV) of the CDx adjusted for this enrichment ± invalid CDx results, showed that the lower bounds of the 95% CIs were all >95% (Table 1). The efficacy OS analysis in the intent-to-treat (ITT) CDx+ population (ITT CDx+=CTA+ & CDx+; N=483: Q, n=242; P, n=241) demonstrated a clinically relevant OS improvement with Q (median OS of 29.4 months) vs P (median OS of 14.8 months), resulting in 14.6 months prolongation of median OS, with an HR of 0.794 (95% CI 0.621-1.014), corresponding to a 20.6% reduction in relative risk of death (Figure 1). Conclusions This study showed 1) agreement between CDx & CTA in identifying nd FLT3-ITD+ AML pts and 2) that OS benefit provided by Q in the ITT CDx+ population is comparable with the OS benefit in the ITT population of Q-F. The LeukoStrat CDx FLT3 Mutation Assay aids in assessing AML pts for Q therapy.
Abstract
Introduction The presence of MRD in patients with AML who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 (FLT3) ...internal tandem duplications (ITD) confer a negative prognostic impact by increasing risk of relapse. The ability to detect FLT3-ITD mutations in remission bone marrow specimens is hampered by the limited sensitivity at 1% of PCR-based assays. To address such limitations, we developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations.
Method Genomic DNA was isolated from bone marrow (BM) aspirates or peripheral blood (PB) samples. PCR was performed to amplify exons 13 to 15 of the FLT3 gene. Highly diverse NGS libraries were then generated and sequenced using Illumina’s sequencer. Using a custom bioinformatics approach, unique FLT3-ITD mutations of varying lengths were identified and mutant allelic frequency calculated. The assay was validated using clinical samples and contrived samples. For accuracy assessment, 30 samples (including PB or BM remnant patient DNA and cell line DNA, above DNA diluted in normal DNA) were included. Data were compared with a Fragment Size Analysis by Capillary Electrophoresis assay. To assess precision, the assay was validated at multiple levels evaluating intra-assay, inter-assay, inter-operator, inter-instrument and inter-reagent lot precision. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity.
Summary The ideal DNA input range was established as 300 ng to 500 ng. In all FLT3-ITD-positive cell line samples covering diverse FLT3-ITD lengths (6 bp to 156 bp), the FLT3-ITD MRD NGS assay showed 100% concordance with the reference assay. The assay is also capable of detecting ITDs as expected after the samples were diluted in normal DNA to mimic samples from patients in remission. All acceptance criteria for the different precision parameters were met. The lower limit of detection of 0.013% regardless of ITD length was established but the assay was capable of detecting FLT3-ITD mutations at a level as low as 0.003% without false-positive results. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% or the lower limit of quantitation.
Conclusion Analytical validation results established the role of this NGS-based MRD assay for the clinical management of FLT3-ITD AML. The FLT3-ITD MRD NGS assay demonstrated high sensitivity and high specificity by detecting the unique length of each patient’s mutation with no false-positive results in expected negative samples. This assay might be helpful in defining the depth of remission, identifying persistent disease, and helping to guide decision making in the use of FLT3 inhibitors as continuation therapy.
Citation Format: Wenge Shi, Christian Laing, Wei Ding, Marc Mycoco, Jelveh Lameh, Reinhold Pollner. Development of a novel next-generation sequencing (NGS)-based assay for measurable residual disease (MRD) in FLT3-ITD acute myeloid leukemia (AML) and its potential clinical application in patients abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2299.
We assessed a simple, noninvasive method of monitoring transcutaneous partial pressure of CO2 (Ptcco2) in mice to determine whether it would provide an accurate and reproducible method to assess ...ventilatory depression in mice. To this end, Ptcco2 and Paco2 (partial pressure of arterial CO2) measurements were performed on isoflurane-anesthetized male C57Bl/6 mice breathing differing percentages of CO2 or fentanyl, a known ventilatory depressive drug. All doses of fentanyl produced a sharp increase in Ptcco2 values within 20 min with difference in Ptcco2 values between saline and all fentanyl groups being statistically significant (P < 0.0001). A good correlation between Paco2 and Ptcco2 values was established (r2 = 0.91). A Bland-Altman analysis likewise found that Ptcco2 measurements in the mice reliably and accurately reflected their Paco2 values. Therefore, under controlled conditions, Ptcco2 measurements were found to reliably reflect Paco2 values in mice. Consequently, the Ptcco2 method can be used as a means to rapidly and quantitatively assess the ventilatory depressive properties of a wide spectrum of drugs, under varying conditions in numerous mouse models.
Abstract
Background: Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene that is a major negative regulator of the Phosphatidylinositol 3-kinase (PI3K) pathway. Loss of PTEN protein ...expression has been mechanistically linked to tumor progression. Blocking the PI3K pathway might inhibit the growth and proliferation of cells that have deletions in PTEN. Thus characterization of PTEN expression in patient tumor samples may assist prediction of potential response to PI3K inhibitor therapies.
Methods: We developed and validated an immunohistochemical assay on Ventana BenchMark XT to detect PTEN in formalin fixed and paraffin-embedded (FFPE) tissue by utilizing a rabbit monoclonal antibody (clone 138G6) from Cell Signaling Technologies that recognizes the carboxy-terminal domain of PTEN. PTEN immunohistochemical staining was performed on 1577 breast tumor specimens to determine PTEN protein loss. A subset of these cases (n=663) was also assessed for PTEN mutation.
Results: Cellular localization of PTEN expression was observed in both the cytoplasm and the nucleus. Both cellular compartments were scored and used in the staining intensity determination. We observed that PTEN staining is sensitive to variation in tissue handling, fixation and antigen retrieval. PTEN staining was affected significantly by antigen retrieval method. Optimal staining conditions were determined to be 1:60 antibody dilution using Citrate pH 6.0 as antigen retrieval buffer. In a cohort of 1577 hormone receptor positive HER2-negative locally advanced or metastatic breast cancer patients, loss of PTEN protein was observed in 8.6% (135/1577) of patients. There was a positive correlation between PTEN mutation rate (17 out of 66 PTEN IHC positive cases vs 14 out of 587 negative cases) and loss of PTEN protein expression. PTEN loss was observed to correlate with better clinical response to PI3K inhibitor.
Conclusions: Pre-analytical handling of samples is important for PTEN IHC staining. PTEN mutations and insertions/deletions contribute to PTEN protein loss. This study validates a simple method to interrogate PTEN status in clinical specimens and supports the utility of this test in selecting patients who are likely to respond to PI3K inhibitor treatment.
Citation Format: Hua Gong, Emmanuel Pacia, Sharmila Manjeshwar, Beiru Chen, Xun Li, Bashar Dabbas, Jelveh Lameh, Naveen Babbar, Shabnam Tangri. Development and characterization of PTEN IHC assay for testing breast cancer patients specimens abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-09-08.
The mechanism by which muscarinic receptors internalize upon agonist exposure is poorly understood. To determine the endocytic pathways responsible for muscarinic receptor internalization, we have ...stably transfected human embryonic kidney (HEK 293) cells with the Hm1 (human muscarinic subtype 1) receptor tagged at the amino terminus with the epitope EYMPME. The subcellular location of the receptor was visualized by immunofluorescence confocal microscopy and quantified with the use of binding studies. The receptor redistributed into intracellular compartments following agonist treatment. This process was reversible upon removal of agonist and inhibited by antagonist. Acid treatment of the cells, which disrupts internalization via clathrin-coated vesicles, inhibited carbachol-stimulated internalization. Phorbol 12-myristate 13-acetate, on the other hand, which inhibits caveolae-mediated endocytosis, had no effect on carbachol-induced endocytosis. Double-labeling confocal microscopy was used to characterize the intracellular vesicles containing Hm1 receptor following agonist treatment. The Hm1 receptor was shown to be colocalized with clathrin and α-adaptin, a subunit of the AP2 adaptor protein which links endocytosed proteins with clathrin in the intracellular vesicles. In addition, endosomes containing Hm1 also contained the transferrin receptor, which internalizes via clathrin-coated vesicles. In contrast, caveolin, the protein that comprises caveolae, did not colocalize with Hm1 in intracellular vesicles following agonist treatment, indicating that caveolae are not involved in the agonist-induced internalization of Hm1. These results indicate that agonist-induced internalization of the Hm1 receptor occurs via clathrin-coated vesicles in HEK cells.
The aim of the present study was to describe the activity of a set of opioid drugs, including partial agonists, in a human embryonic kidney cell system stably expressing only the mouse kappa-opioid ...receptors. Receptor activation was assessed by measuring the inhibition of cyclic adenosine mono phosphate (cAMP) production stimulated by 5 microM forskolin. Intrinsic activities and potencies of these ligands were determined relative to the endogenous ligand dynorphin and the kappa agonist with the highest intrinsic activity that was identified in this study, fentanyl.
Among the ligands studied naltrexone, WIN 44,441 and dezocine, were classified as antagonists, while the remaining ligands were agonists. Intrinsic activity of agonists was assessed by determining the extent of inhibition of forskolin-stimulated cAMP production. The absolute levels of inhibition of cAMP production by each ligand was used to describe the rank order of intrinsic activity of the agonists; fentanyl = lofentanil > or = hydromorphone = morphine = nalorphine > or = etorphine > or = xorphanol > or = metazocine > or = SKF 10047 = cyclazocine > or = butorphanol > nalbuphine. The rank order of affinity of these ligands was; cyclazocine > naltrexone > or = SKF 10047 > or = xorphanol > or = WIN 44,441 > nalorphine > butorphanol > nalbuphine > or = lofentanil > dezocine > or = metazocine > or = morphine > hydromorphone > fentanyl.
These results elucidate the relative activities of a set of opioid ligands at kappa-opioid receptor and can serve as the initial step in a systematic study leading to understanding of the mode of action of these opioid ligands at this receptor.
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