ABSTRACT
Six new ravidomycin analogs (1-4, 6, and 7) were isolated from Streptomyces sp. Am59 using UV- and LCMS-guided separation based on Global Natural Products Social (GNPS) molecular networking analysis. Furthermore, we isolated fucomycin V (9), which possesses the same chromophore as ravidomycin but features a d-fucopyranose instead of d-ravidosamine. This is the first report of 9 as a natural product. Four new analogs (10-13) of 9 were also isolated. The structures were elucidated by combined spectroscopic and computational methods. We also found an inconsistency with the published [α]D25 of deacetylravidomycin, which is reported to have a (-) sign. Instead, we observed a (+) specific rotation for the reported absolute configuration of deacetylravidomycin (containing d-ravidosamine). We confirmed the positive sign by reisolating deacetylravidomycin from S. ravidus and by deacetylating ravidomycin. Finally, antibacterial, antifungal, and cytotoxicity activities were determined for the compounds. Compared to deacetylravidomycin, the compounds 4-6, 9, 11, and 12 exhibited greater antibacterial selectivity.
Subject(s)
Antineoplastic Agents , Streptomyces , Streptomyces/chemistry , Aminoglycosides , Anti-Bacterial Agents/chemistry , Molecular StructureABSTRACT
Introduction: Approximately 50% of melanomas harbor an activating BRAFV600E mutation. Standard of care involves a combination of inhibitors targeting mutant BRAF and MEK1/2, the substrate for BRAF in the MAPK pathway. PTEN loss-of-function mutations occur in ~40% of BRAFV600E melanomas, resulting in increased PI3K/AKT activity that enhances resistance to BRAF/MEK combination inhibitor therapy. Methods: To compare the response of PTEN null to PTEN wild-type cells in an isogenic background, CRISPR/Cas9 was used to knock out PTEN in a melanoma cell line that harbors a BRAFV600E mutation. RNA sequencing, functional kinome analysis, and drug synergy screening were employed in the context of BRAF/MEK inhibition. Results: RNA sequencing and functional kinome analysis revealed that the loss of PTEN led to an induction of FOXD3 and an increase in expression of the FOXD3 target gene, ERBB3/HER3. Inhibition of BRAF and MEK1/2 in PTEN null, BRAFV600E cells dramatically induced the expression of ERBB3/HER3 relative to wild-type cells. A synergy screen of epigenetic modifiers and kinase inhibitors in combination with BRAFi/MEKi revealed that the pan ERBB/HER inhibitor, neratinib, could reverse the resistance observed in PTEN null, BRAFV600E cells. Conclusions: The findings indicate that PTEN null BRAFV600E melanoma exhibits increased reliance on ERBB/HER signaling when treated with clinically approved BRAFi/MEKi combinations. Future studies are warranted to test neratinib reversal of BRAFi/MEKi resistance in patient melanomas expressing ERBB3/HER3 in combination with its dimerization partner ERBB2/HER2.
ABSTRACT
The tumor suppressor, death-associated protein kinase (DAPK), is a Ca(2+)/calmodulin-regulated Ser/Thr kinase with an important role in regulating cytoskeletal dynamics. Autophosphorylation within the calmodulin-binding domain at Ser-308 inhibits DAPK catalytic activity. Dephosphorylation of Ser-308 by a previously unknown phosphatase enhances kinase activity and proteasome-mediated degradation of DAPK. In these studies, we identified two holoenzyme forms of protein phosphatase 2A (PP2A), ABalphaC and ABdeltaC, as DAPK-interacting proteins. These phosphatase holoenzymes dephosphorylate DAPK at Ser-308 in vitro and in vivo resulting in enhanced kinase activity of DAPK. The enzymatic activity of PP2A also negatively regulates DAPK levels by enhancing proteasome-mediated degradation of the kinase. Overexpression of wild type DAPK induces cell rounding and detachment in HEK293 cells; however, this effect is not observed following expression of an inactive DAPK S308E mutant. Finally, activation of DAPK by PP2A was found to be required for ceramide-induced anoikis. Together, our results provide a mechanism by which PP2A and DAPK activities control cell adhesion and anoikis.
Subject(s)
Anoikis/drug effects , Ceramides/pharmacology , Protein Phosphatase 2/metabolism , Amino Acid Substitution , Anoikis/genetics , Apoptosis Regulatory Proteins , Calcium-Calmodulin-Dependent Protein Kinases , Cell Adhesion/drug effects , Cell Adhesion/genetics , Cytoskeleton/genetics , Cytoskeleton/metabolism , Death-Associated Protein Kinases , HeLa Cells , Holoenzymes/genetics , Holoenzymes/metabolism , Humans , Mutation, Missense , Phosphorylation/drug effects , Phosphorylation/genetics , Protein Phosphatase 2/genetics , Tumor Suppressor ProteinsABSTRACT
Dysregulation of PI3K/Akt signaling is a dominant feature in basal-like or triple-negative breast cancers (TNBC). However, the mechanisms regulating this pathway are largely unknown in this subset of aggressive tumors. Here we demonstrate that the transcription factor SOX4 is a key regulator of PI3K signaling in TNBC. Genomic and proteomic analyses coupled with mechanistic studies identified TGFBR2 as a direct transcriptional target of SOX4 and demonstrated that TGFBR2 is required to mediate SOX4-dependent PI3K signaling. We further report that SOX4 and the SWI/SNF ATPase SMARCA4, which are uniformly overexpressed in basal-like tumors, form a previously unreported complex that is required to maintain an open chromatin conformation at the TGFBR2 regulatory regions in order to mediate TGFBR2 expression and PI3K signaling. Collectively, our findings delineate the mechanism by which SOX4 and SMARCA4 cooperatively regulate PI3K/Akt signaling and suggest that this complex may play an essential role in TNBC genesis and/or progression.
ABSTRACT
Neurofibromatosis Type 2 (NF2) is an autosomal dominant genetic syndrome caused by mutations in the NF2 tumor suppressor gene resulting in multiple schwannomas and meningiomas. There are no FDA approved therapies for these tumors and their relentless progression results in high rates of morbidity and mortality. Through a combination of high throughput screens, preclinical in vivo modeling, and evaluation of the kinome en masse, we identified actionable drug targets and efficacious experimental therapeutics for the treatment of NF2 related schwannomas and meningiomas. These efforts identified brigatinib (ALUNBRIG®), an FDA-approved inhibitor of multiple tyrosine kinases including ALK, to be a potent inhibitor of tumor growth in established NF2 deficient xenograft meningiomas and a genetically engineered murine model of spontaneous NF2 schwannomas. Surprisingly, neither meningioma nor schwannoma cells express ALK. Instead, we demonstrate that brigatinib inhibited multiple tyrosine kinases, including EphA2, Fer and focal adhesion kinase 1 (FAK1). These data demonstrate the power of the de novo unbiased approach for drug discovery and represents a major step forward in the advancement of therapeutics for the treatment of NF2 related malignancies.
Subject(s)
Meningeal Neoplasms/genetics , Meningioma/genetics , Neurilemmoma/genetics , Neurofibromin 2/deficiency , Neurofibromin 2/genetics , Organophosphorus Compounds/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Cell Proliferation , Humans , Mutation , Neurilemmoma/pathologyABSTRACT
To better understand the distinct functional roles of the 220- and 130-kDa forms of myosin light chain kinase (MLCK), expression and intracellular localization were determined during development and in adult mouse tissues. Northern blot, Western blot, and histochemical studies show that the 220-kDa MLCK is widely expressed during development as well as in several adult smooth muscle and nonmuscle tissues. The 130-kDa MLCK is highly expressed in all adult tissues examined and is also detectable during embryonic development. Colocalization studies examining the distribution of 130- and 220-kDa mouse MLCKs revealed that the 130-kDa MLCK colocalizes with nonmuscle myosin IIA but not with myosin IIB or F-actin. In contrast, the 220-kDa MLCK did not colocalize with either nonmuscle myosin II isoform but instead colocalizes with thick interconnected bundles of F-actin. These results suggest that in vivo, the physiological functions of the 220- and 130-kDa MLCKs are likely to be regulated by their intracellular trafficking and distribution.