Publications

Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that the RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC-overexpressing mouse models and patient-derived xenografts. MSI1 inhibition abrogates tumor initiation and significantly prolongs survival in both models. We identify binding targets of MSI1 in normal neural and G3 MB stem cells and then cross referenced these data with unbiased large-scale screens at the transcriptomic, translatomic and proteomic levels to systematically dissect its functional role. Comparative integrative multi-omic analyses of these large datasets reveal cancer-selective MSI1-bound targets sharing multiple MYC associated pathways, providing a valuable resource for context-specific therapeutic targeting of G3 MB.

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We report a concise synthesis of the naturally occurring protein synthesis inhibitor (+)-actinobolin (1). The densely functionalized and stereochemically complex molecular structure of 1 was assembled from (−)-quinic acid, L-threonine, and L-alanine as the principal components. Our route is based around a convergent strategy that features conjugate addition of an α-amino radical in the key fragment-coupling step. The dramatically simplified synthesis of (+)-actinobolin proceeding in 9 steps with 18 % overall yield has practical implications for analog preparation, as demonstrated herein.

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RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron via an ordered assembly of intermediate complexes. Spliceosome activation is a major control step requiring dramatic protein and RNA rearrangements leading to a catalytically active complex. Splicing factor 3B subunit 1 (SF3B1) protein, a subunit of the U2 snRNP, is phosphorylated during spliceosome activation, but the responsible kinase has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N-terminus during spliceosome activation. The phosphorylation is important for association of SF3B1 with U5 and U6 snRNAs in spliceosome activated B^act complex and it can be blocked by OTS964, a potent and highly selective inhibitor of CDK11. CDK11 inhibition prevents spliceosomal transition from the precatalytic complex B to the activated complex B^act and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNA and chromatin. We characterize OTS964 as a quality chemical biology probe for CDK11 and demonstrate a central role of CDK11 in spliceosome assembly and regulation of splicing.

Bilirubin (BR) is an essential metabolite formed by the catabolism of heme. Phototherapy with blue-green light can be applied to reduce high concentrations of BR in blood and is used especially in the neonatal period. In this work, we studied the photochemistry of (Z)-isovinylneoxanthobilirubic acid methyl ester, a dipyrrinone subunit of BR, by steady-state absorption, femtosecond transient absorption, and stimulated Raman spectroscopies. Both the (Z)- and (E)-configurational isomers of isovinylneoxanthobilirubic acid undergo wavelength-dependent and reversible photoisomerization. The isomerization from the excited singlet state is ultrafast (the lifetimes of (Z)- and (E)-isomers were found to be ∼0.9 and 0.1 ps, respectively), and its efficiencies increase with increased photon energy. In addition, we studied sensitized photooxidation of the dipyrrinone subunit by singlet oxygen that leads to the formation of propentdyopents. Biological activities of these compounds, namely, effects on the superoxide production, lipoperoxidation, and tricarboxylic acid cycle metabolism, were also studied. Finally, different photochemical and biological properties of this BR subunit and its structural analogue, (Z)-vinylneoxanthobilirubic acid methyl ester, studied before, are discussed.

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Histone methyltransferase DOT1L is an attractive therapeutic target for treatment of hematological malignancies. Herein, we report design, synthesis, and profiling of new DOT1L inhibitors based on non-routine carbocyclic C-nucleoside scaffold. The experimentally observed SAR was found to be non-trivial as seemingly minor changes of individual substituents resulted in significant changes in the affinity to DOT1L. Molecular modeling suggested that these trends could be related to significant conformational changes of the protein upon interaction with the inhibitors. The compounds 22 and (-)-53 (MU1656), carbocyclic C-nucleoside analogs of the natural nucleoside derivative EPZ004777 and the clinical candidate EPZ5676 (pinometostat), potently and selectively inhibit DOT1L in vitro as well as in the cell. The most potent compound MU1656 was found to be more metabolically stable and significantly less toxic in vivo than pinometostat itself.

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Many dynamic interactions within the cell microenvironment modulate cell behavior and cell fate. However, the pathways and mechanisms behind cell–cell or cell–extracellular matrix interactions remain understudied, as they occur at a nanoscale level. Recent progress in nanotechnology allows for mimicking of the microenvironment at nanoscale in vitro; electron-beam lithography (EBL) is currently the most promising technique. Although this nanopatterning technique can generate nanostructures of good quality and resolution, it has resulted, thus far, in the production of only simple shapes (e.g., rectangles) over a relatively small area (100 × 100 μm), leaving its potential in biological applications unfulfilled. Here, we used EBL for cell-interaction studies by coating cell-culture-relevant material with electron-conductive indium tin oxide, which formed nanopatterns of complex nanohexagonal structures over a large area (500 × 500 μm). We confirmed the potential of EBL for use in cell-interaction studies by analyzing specific cell responses toward differentially distributed nanohexagons spaced at 1000, 500, and 250 nm. We found that our optimized technique of EBL with HaloTags enabled the investigation of broad changes to a cell-culture-relevant surface and can provide an understanding of cellular signaling mechanisms at a single-molecule level.

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Tissue clearing methods eliminate the need for sectioning, thereby helping better understand the 3D organization of tissues and organs. In the past fifteen years, clearing methods have been developed to preserve endogenous fluorescent protein tags. Some of these methods (ClearSee, TDE, PEA-Clarity, etc.) were adapted to clear various plant species, with the focus on roots, leaves, shoot apical meristems, and floral parts. However, these methods have not been used in developing seeds beyond the early globular stage. Tissue clearing is problematic in post-globular seeds due to various apoplastic barriers and secondary metabolites. In this study, we compared six methods for their efficiency in clearing Arabidopsis thaliana seeds at post-globular embryonic stages. Three methods (TDE, ClearSee, and ClearSee alpha) have already been reported in plants, whereas the others (fsDISCO, FAST9, and CHAPS clear) are used in this context for the first time. These methods were assessed for seed morphological changes, clearing capacity, removal of tannins, and spectral properties. We tested each method in seeds from globular to mature stages. The pros and cons of each method are listed herein. ClearSee alpha appears to be the method of choice as it preserves seed morphology and prevents tannin oxidation. However, FAST9 with 60% iohexol as a mounting medium is faster, clears better, and appears suitable for embryonic shape imaging. Our results may guide plant researchers to choose a suitable method for imaging fluorescent protein-labeled embryos in intact Arabidopsis seeds.

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