19Fluorine-MRI Based Longitudinal Immuno-Microenvironment-Monitoring for Pancreatic Cancer.

BACKGROUND: Pancreatic cancer has a poor prognosis. Targeting Kirsten Rat Sarcoma (KRAS) mutation and its related pathways may enhance immunotherapy efficacy. While in vivo monitoring of therapeutic response and immune cell migration remains challenging, Fluorine-19 MRI (19F MRI) may allow noninvasive longitudinal imaging of immune cells. PURPOSE: Evaluating the potential of 19F MRI for monitoring changes in the tumor immune microenvironment, in response to combined SHP2/MEK inhibition. STUDY TYPE: Pre-clinical animal study. ANIMAL MODEL: Murine genetically engineered pancreatic cancer model (N = 20, both sexes). FIELD STRENGTH/SEQUENCE: 9.4-T, two-dimensional multi-slice Rapid Acquisition with Relaxation Enhancement sequence. Intravenous injection of 19F-perfluorocarbon (PFC) nanoparticles. ASSESSMENT: Upon tumor detection by conventional 1H MRI screening, 19F MRI was performed in mice 24 hours after PFC nanoparticle administration. Animals were randomly assigned to four treatment groups: allosteric Src-homology-2-containing protein tyrosine phosphatase 2 (SHP2) inhibitor SHP099, the mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) inhibitor Trametinib, the combination of both, or a vehicle control (4 to 6 mice each group), administered every other day per oral gavage. 1H and 19F MRI was repeated 7 days and 14 days later. Pancreatic immune cell infiltrates were analyzed by flow cytometry and multiplex immunohistofluorescence (mIHF) upon sacrifice. STATISTICAL TESTS: Independent t-tests and one-way analysis of variance. RESULTS: 19F MRI revealed continuous decrease of PFC-signals in tumors from vehicle controls (100%, 80%, and 74% on days 0, 7, and 14, respectively), contrasting with stable or increasing signals under KRAS-pathway-directed treatment. MEK inhibition showed 100%, 152%, and 84% and dual SHP2/MEK1/2 inhibition demonstrated signals of 100%, 134%, and 100% on days 0, 7, 14, respectively. mIHF analyses indicated CD11b+ macrophages/monocytes as primary contributors to the observed 19F MRI signal differences. DATA CONCLUSION: 19F MRI might provide non-invasive longitudinal estimates for abundance and spatial distribution of CD11b+ macrophages/monocytes in pancreatic cancer. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 2.

Tyrosine phosphatase PTPN11/SHP2 in solid tumors - bull's eye for targeted therapy?

Encoded by PTPN11, the Src-homology 2 domain-containing phosphatase 2 (SHP2) integrates signals from various membrane-bound receptors such as receptor tyrosine kinases (RTKs), cytokine and integrin receptors and thereby promotes cell survival and proliferation. Activating mutations in the PTPN11 gene may trigger signaling pathways leading to the development of hematological malignancies, but are rarely found in solid tumors. Yet, aberrant SHP2 expression or activation has implications in the development, progression and metastasis of many solid tumor entities. SHP2 is involved in multiple signaling cascades, including the RAS-RAF-MEK-ERK-, PI3K-AKT-, JAK-STAT- and PD-L1/PD-1- pathways. Although not mutated, activation or functional requirement of SHP2 appears to play a relevant and context-dependent dichotomous role. This mostly tumor-promoting and infrequently tumor-suppressive role exists in many cancers such as gastrointestinal tumors, pancreatic, liver and lung cancer, gynecological entities, head and neck cancers, prostate cancer, glioblastoma and melanoma. Recent studies have identified SHP2 as a potential biomarker for the prognosis of some solid tumors. Based on promising preclinical work and the advent of orally available allosteric SHP2-inhibitors early clinical trials are currently investigating SHP2-directed approaches in various solid tumors, either as a single agent or in combination regimes. We here provide a brief overview of the molecular functions of SHP2 and collate current knowledge with regard to the significance of SHP2 expression and function in different solid tumor entities, including cells in their microenvironment, immune escape and therapy resistance. In the context of the present landscape of clinical trials with allosteric SHP2-inhibitors we discuss the multitude of opportunities but also limitations of a strategy targeting this non-receptor protein tyrosine phosphatase for treatment of solid tumors.

The ubiquitin-like modifier FAT10 is degraded by the 20S proteasome in vitro but not in cellulo.

Ubiquitin-independent protein degradation via the 20S proteasome without the 19S regulatory particle has gained increasing attention over the last years. The degradation of the ubiquitin-like modifier FAT10 by the 20S proteasome was investigated in this study. We found that FAT10 was rapidly degraded by purified 20S proteasomes in vitro, which was attributed to the weak folding of FAT10 and the N-terminally disordered tail. To confirm our results in cellulo, we established an inducible RNA interference system in which the AAA-ATPase Rpt2 of the 19S regulatory particle is knocked down to impair the function of the 26S proteasome. Using this system, degradation of FAT10 in cellulo was strongly dependent on functional 26S proteasome. Our data indicate that in vitro degradation studies with purified proteins do not necessarily reflect biological degradation mechanisms occurring in cells and, therefore, cautious data interpretation is required when 20S proteasome function is studied in vitro.

Abundance and localization of human UBE3A protein isoforms.

Loss of UBE3A expression, a gene regulated by genomic imprinting, causes Angelman syndrome (AS), a rare neurodevelopmental disorder. The UBE3A gene encodes an E3 ubiquitin ligase with three known protein isoforms in humans. Studies in mouse suggest that the human isoforms may have differences in localization and neuronal function. A recent case study reported mild AS phenotypes in individuals lacking one specific isoform. Here we have used CRISPR/Cas9 to generate isogenic human embryonic stem cells (hESCs) that lack the individual protein isoforms. We demonstrate that isoform 1 accounts for the majority of UBE3A protein in hESCs and neurons. We also show that UBE3A predominantly localizes to the cytoplasm in both wild type and isoform-null cells. Finally, we show that neurons lacking isoform 1 display a less severe electrophysiological AS phenotype.