Melanomas arising in the mucous membranes are a rare and aggressive subtype. New treatment approaches are needed, yet accumulating sufficient evidence to improve patient outcomes is difficult. Clinical and pathological correlates between human and canine mucosal melanomas (MM) are substantial, and the relatively greater incidence of spontaneous naturally occurring MM in dogs represents a promising opportunity for predictive modeling. The genomic landscapes of human and canine MM appear highly diverse and generally lack recurring hotspot mutations associated with cutaneous melanomas. Although much remains to be determined, evidence indicates that Ras/MAPK and/or PI3K/Akt/mTOR signaling pathway activations are common in both species and may represent targets for therapeutic intervention. Sapanisertib, an mTORC1/2 inhibitor, was selected from a PI3K/mTOR inhibitor library to collaborate with MEK inhibition; the latter preclinical efficacy was demonstrated previously for canine MM. Combined inhibition of MEK and mTORC1/2, using trametinib and sapanisertib, produced apoptosis and cell cycle alteration, synergistically reducing cell survival in canine MM cell lines with varying basal signaling activation levels. Compared to individual inhibitors, a staggered sapanisertib dose, coupled with daily trametinib, was optimal for limiting primary MM xenograft growth in mice, and tumor dissemination in a metastasis model, while minimizing hematologic and renal side effects. Inhibitors downmodulated respective signaling targets and the combination additionally suppressed pathway reciprocal crosstalk. The combination did not significantly change plasma sapanisertib pharmacokinetics, however trametinib area under the curve was increased in the presence of sapanisertib. Targeting Ras/MAPK and PI3K/Akt/mTOR signal transduction pathways appear rational therapies for canine and human MM.

Remdesivir, formerly GS-5734, has recently become the first antiviral drug approved by the U.S. Food and Drug Administration (FDA) to treat COVID-19, the disease caused by SARS-CoV-2. Therapeutic dosing and pharmacokinetic studies require a simple, sensitive, and selective validated assay to quantify drug concentrations in clinical samples. Therefore, we developed a rapid and sensitive LC-MS/MS assay for the quantification of remdesivir in human plasma with its deuterium-labeled analog, remdesivir- 2 H5, as the internal standard. Chromatographic separation was achieved on a Phenomenex® Synergi TM HPLC Fusion-RP (100 x 2 mm, 4 μm) column by gradient elution. Excellent accuracy and precision (<5.2% within-run variations and <9.8% between-run variations) were obtained over the range of 0.5-5000 ng/mL. The assay met the FDA Bioanalytical Guidelines for selectivity and specificity, and low inter-matrix lot variability (<2.7%) was observed for extraction efficiency (77%) and matrix effect (123%) studies. Further, stability tests showed that the analyte does not degrade under working conditions, nor during freezing and thawing processes.

Breast cancer metastasis is a leading cause of cancer-related death of women in the United States. A hurdle in advancing metastasis-targeted intervention is the phenotypic heterogeneity between primary and secondary lesions. To identify metastasisspecific gene expression profiles we performed RNA-sequencing of breast cancer mouse models; analyzing metastases from models of various drivers and routes. We contrasted the models and identified common, targetable signatures. Allograft models exhibited more mesenchymal-like gene expression than genetically engineered mouse models (GEMM), and primary culturing of GEMM-derived metastatic tissue induced mesenchymal-like gene expression. In addition, metastasis-specific transcriptomes differed between tail vein and orthotopic injection of the same cell line. Gene expression common to models of spontaneous metastasis included sildenafil response and nicotine degradation pathways. Strikingly, in vivo sildenafil treatment significantly reduced metastasis by 54%, while nicotine significantly increased metastasis by 46%. These data suggest that (i) actionable metastasis-specific pathways can be readily identified, (ii) already available drugs may have great potential to alleviate metastatic incidence, and (iii) metastasis may be influenced greatly by lifestyle choices such as the choice to consume nicotine products. In summary, while mouse models of breast cancer metastasis vary in ways that must not be ignored, there are shared features that can be identified and potentially targeted therapeutically. Implications: The data we present here exposes critical variances between preclinical models of metastatic breast cancer and identifies targetable pathways integral to metastatic spread.

mining cellular viability. Taken together, in our current study we have developed a PEGylated targeted nanoconjugate ACG44P1000 that showed enhanced selectivity towards pancreatic cancer cells and pancreatic stellate cells, among others, for gemcitabine delivery. We will investigate the ability of these optimized conjugates to inhibit desmoplasia and tumor growth in vivo in our future studies.

The blood-tumor barrier (BTB) limits the entry of effective chemotherapeutic agents into the brain for treatment of malignant tumors like glioblastoma (GBM). Poor drug entry across the BTB allows infiltrative glioma stem cells (GSC) to evade therapy and develop treatment resistance. Regadenoson, an FDA-approved adenosine A2A receptor (A2AR) agonist, has been shown to increase drug delivery across the blood-brain barrier (BBB) in non-tumor bearing rodents without a defined mechanism of enhancing BTB permeability. Here, we characterize the time-dependent impact of regadenoson on brain endothelial cell interactions and paracellular transport, using mouse and rat brain endothelial cells and tumor models. In vitro, A2AR activation leads to disorganization of cytoskeletal actin filaments by 30 minutes, down-regulation of junctional protein expression by 4 hours, and re-establishment of endothelial cell integrity by 8 hours. In rats bearing intracranial gliomas, regadenoson treatment results in increase of intratumoral temozolomide (TMZ) concentrations, yet no increased survival noted with combined TMZ therapy. These findings demonstrate regadenoson's ability to induce brain endothelial structural changes amongst glioma to increase BTB permeability. The use of vasoactive mediators, like regadenoson, which transiently influences paracellular transport, should further be explored to evaluate their potential to enhance CNS treatment delivery to aggressive brain tumors. ImplicationsThis study provides insight on the use of a vasoactive agent to increase exposure of the bloodtumor barrier to chemotherapy with intention to improve glioma treatment efficacy.