Pharmacologically targeting the HER2 oncoprotein with therapeutics such as the mAb, trastuzumab, provides clinical benefit for patients with HER2-positive (HER2+) cancers. However, a significant ...number of patients eventually progress on these therapies. Efforts to overcome therapeutic resistance through combination therapy with small-molecule inhibitors of HER2 have been limited by toxicities associated with off-target activity and/or limited efficacy. In this preclinical study, we explore single-agent and combined activity of tucatinib, a novel HER2-selective small-molecule inhibitor. Tucatinib demonstrated potent, selective activity in a panel of 456 human cancer cell lines, with activity restricted to cell lines (breast and non-breast) with HER2-amplification, including models of acquired resistance to trastuzumab. Within the HER2+ population, tucatinib response tracked strongly with HER2-driven signaling. Single-agent tucatinib induced tumor regressions in xenograft models of HER2+ breast cancer and combination with trastuzumab induced a complete and sustained blockade of HER2/PI3K/AKT signaling. Efficacy of the tucatinib/trastuzumab combination matched that induced by current standard-of-care trastuzumab/pertuzumab/docetaxel combination, with the exception that the chemotherapy-sparing tucatinib/trastuzumab combination did not require a dosing holiday to achieve the same efficacy. In xenograft models of HER2+ breast cancer that also express estrogen receptor (ER; HER2+/ER+), tucatinib showed combined efficacy with inhibitors of CDK4/6 and ER, indicating potential novel therapeutic strategies for difficult-to-treat subtypes of HER2+ breast cancer. These data support expanded clinical investigations of tucatinib as a combination partner for other novel and approved targeted therapies for HER2-driven malignancies.
Dentate granule cell (DGC) neurogenesis persists throughout life in the mammalian hippocampal dentate gyrus and increases after epileptogenic insults. The DGC layer in human and experimental mesial ...temporal lobe epilepsy (mTLE) often shows abnormal dispersion and the appearance of hilar-ectopic DGCs. In the pilocarpine mTLE model, hilar-ectopic DGCs arise as a result of an aberrant chain migration of neural progenitors. Reelin is a secreted migration guidance cue that persists in the adult rodent and human hippocampus. We tested the hypothesis that loss of Reelin in the epileptic dentate gyrus leads to aberrant chain migration of DGC precursors. We found that interneuron subsets typically lost in human and experimental mTLE express Reelin, and DGC progenitors express the downstream Reelin signaling molecule Disabled 1 (Dab1). Prolonged seizures decreased Reelin immunoreactivity in the adult rat dentate gyrus and increased Dab1 expression in hilar-ectopic neuroblasts. Exogenous Reelin increased detachment of chain-migrating neuroblasts in dentate gyrus explants, and blockade of Reelin signaling increased chain migration. These findings suggest that Reelin modulates DGC progenitor migration to maintain normal DGC integration in the neonatal and adult mammalian dentate gyrus. Loss of Reelin expression in the epileptic adult hippocampus, moreover, likely contributes to ectopic chain migration and aberrant integration of newborn DGCs.
A special tryptic digestion method has been developed to facilitate rapid identification and accurate quantification of site-specific aspartyl succinimide (Asu) formation in complex protein ...molecules, such as monoclonal antibodies (mAbs). This method replaces chaotropic reagents, such as urea and guanidine hydrochloride (GdnHCl) with an acid labile surfactant RapiGest (RG), eliminates alkylation and desalting steps, and accomplishes the reduced tryptic digestion of an IgG2 mAb in a mildly acidic condition (pH 6.0) with half the time required by conventional methods. The new digestion condition preserves the labile Asu during sample preparation and solves the problem that conventional method has been facing in detecting and quantifying Asu in complex proteins. The validity of this method was confirmed by subjecting a mixture of peptides containing a predetermined amount of Asu to the same digestion conditions. An excellent correlation was also observed for the Asu results from cation-exchange chromatography (CEX) and tryptic peptide maps generated with the new digestion method. This method is also applicable to other enzymatic digestions and used to monitor site-specific deamidation, isomerization, and other chemical modifications in complex proteins by LC/MS.
The Fc region has two highly conserved methionine residues, Met 33 (CH2 domain) and Met 209 (CH3 domain), which are important for the Fc’s structure and biological function. To understand the effect ...of methionine oxidation on the structure and stability of the human IgG1 Fc expressed in Escherichia coli, we have characterized the fully oxidized Fc using biophysical (DSC, CD, and NMR) and bioanalytical (SEC and RP-HPLC−MS) methods. Methionine oxidation resulted in a detectable secondary and tertiary structural alteration measured by circular dichroism. This is further supported by the NMR data. The HSQC spectral changes indicate the structures of both CH2 and CH3 domains are affected by methionine oxidation. The melting temperature (T m) of the CH2 domain of the human IgG1 Fc was significantly reduced upon methionine oxidation, while the melting temperature of the CH3 domain was only affected slightly. The change in the CH2 domain T m depended on the extent of oxidation of both Met 33 and Met 209. This was confirmed by DSC analysis of methionine-oxidized samples of two site specific methionine mutants. When incubated at 45 °C, the oxidized Fc exhibited an increased aggregation rate. In addition, the oxidized Fc displayed an increased deamidation (at pH 7.4) rate at the Asn 67 and Asn 96 sites, both located on the CH2 domain, while the deamidation rates of the other residues were not affected. The methionine oxidation resulted in changes in the structure and stability of the Fc, which are primarily localized to the CH2 domain. These changes can impact the Fc’s physical and covalent stability and potentially its biological functions; therefore, it is critical to monitor and control methionine oxidation during manufacturing and storage of protein therapeutics.
As members of the proneural basic-helix-loop-helix (bHLH) family of transcription factors, Ascl1 and Neurog2 direct the differentiation of specific populations of neurons at various times and ...locations within the developing nervous system. In order to characterize the mechanisms employed by these two bHLH factors, we generated stable, doxycycline-inducible lines of P19 embryonic carcinoma cells that express comparable levels of
Ascl1
and
Neurog2
. Upon induction, both Ascl1 and Neurog2 directed morphological and immunocytochemical changes consistent with initiation of neuronal differentiation. Comparison of Ascl1- and Neurog2-regulated genes by microarray analyses showed both shared and distinct transcriptional changes for each bHLH protein. In both Ascl1- and Neurog2-differentiating cells, repression of Oct4 mRNA levels was accompanied by increased Oct4 promoter methylation. However, DNA demethylation was not detected for genes induced by either bHLH protein. Neurog2-induced genes included glutamatergic marker genes while Ascl1-induced genes included GABAergic marker genes. The Neurog2-specific induction of a gene encoding a protein phosphatase inhibitor,
Ppp1r14a
, was dependent on distinct, canonical E-box sequences within the
Ppp1r14a
promoter and the nucleotide sequences within these E-boxes were partially responsible for Neurog2-specific regulation. Our results illustrate multiple novel mechanisms by which Ascl1 and Neurog2 regulate gene repression during neuronal differentiation in P19 cells.
J. Neurochem (2012) 120, 667–683.
cAMP‐dependent protein kinase (PKA) plays a critical role in nervous system development by modulating sonic hedgehog and bone morphogenetic protein signaling. In the ...current studies, P19 embryonic carcinoma cells were neuronally differentiated by expression of the proneural basic helix‐loop‐helix transcription factor Ascl1. After expression of Ascl1, but prior to expression of neuronal markers such as microtubule associated protein 2 and neuronal β‐tubulin, P19 cells demonstrated a large, transient increase in both mRNA and protein for the endogenous protein kinase inhibitor (PKI)β. PKIβ‐targeted shRNA constructs both reduced the levels of PKIβ expression and blocked the neuronal differentiation of P19 cells. This inhibition of differentiation was rescued by transfection of a shRNA‐resistant expression vector for the PKIβ protein, and this rescue required the PKA‐specific inhibitory sequence of the PKIβ protein. PKIβ played a very specific role in the Ascl1‐mediated differentiation process as other PKI isoforms were unable to rescue the deficit conferred by shRNA‐mediated knockdown of PKIβ. Our results define a novel requirement for PKIβ and its inhibition of PKA during neuronal differentiation of P19 cells.
Abstract only Introduction: Language barriers between acute ischemic stroke patients and clinicians can create challenges in use of intravenous thrombolysis (IVT) as successful administration hinges ...on quick information gathering, shared decision making and consent with the patient and family. Our objectives were to compare IVT door-to-needle (DTN) times and consent between English and non-English speaking patients. Methods: We conducted a single institution retrospective chart analysis from 2018-2023 via manual data extraction of the electronic medical record to determine any disparities in quality metrics between English and non-English speakers treated with IVT. Standard time-based metrics were pulled from the AHA Get with the Guidelines registry. We included adult patients treated onsite, excluding telestroke encounters. We used standard descriptive statistics and a 2-paired t-test to compare treatment times. Results: We included 215 English speakers and 19 non-English speakers in our analysis. Consent was obtained in 92% (197/215) of English speakers and 90% (17/19) of non-English speakers, the remaining implied consent or consent was undocumented. Mean DTN times were 48.8 min and 48.2 min for English and non-English speakers, respectively, and differences were not statistically significant. Discussion: Previous studies have reported lower IVT use, longer DTN times and poorer clinical outcomes in patients who required an interpreter. However, others reported no difference in DTN times in non-English patients, attributing availability of translating services and diversity of healthcare providers. In our population, DTN times remained equally fast, but we remain unsure if our non-English speakers are appropriately consented, understanding their acute care. Future directions include improving consent documentation, and conducting qualitative interviews of English and non-English speakers who receive IVT. Our ultimate goal is to ensure quality of care for this vulnerable population.
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