Selective Pharmacological Targeting of a DEAD Box RNA Helicase

RNA helicases represent a large family of proteins implicated in many biological processes including ribosome biogenesis, splicing, translation and mRNA degradation. However, these proteins have little substrate specificity, making inhibition of selected helicases a challenging problem. The prototypical DEAD box RNA helicase, eIF4A, works in conjunction with other translation factors to prepare mRNA templates for ribosome recruitment during translation initiation. Herein, we provide insight into the selectivity of a small molecule inhibitor of eIF4A, hippuristanol. This coral-derived natural product binds to amino acids adjacent to, and overlapping with, two conserved motifs present in the carboxy-terminal domain of eIF4A. Mutagenesis of amino acids within this region allowed us to alter the hippuristanol-sensitivity of eIF4A and undertake structure/function studies. Our results provide an understanding into how selective targeting of RNA helicases for pharmacological intervention can be achieved

Antitumor Activity and Mechanism of Action of the Cyclopenta[b]benzofuran, Silvestrol

BACKGROUND. Flavaglines are a family of natural products from the genus Aglaia that exhibit anti-cancer activity in vitro and in vivo and inhibit translation initiation. They have been shown to modulate the activity of eIF4A, the DEAD-box RNA helicase subunit of the eukaryotic initiation factor (eIF) 4F complex, a complex that stimulates ribosome recruitment during translation initiation. One flavagline, silvestrol, is capable of modulating chemosensitivity in a mechanism-based mouse model. METHODOLOGY/PRINCIPAL FINDINGS. Among a number of flavagline family members tested herein, we find that silvestrol is the more potent translation inhibitor among these. We find that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. We show that silvestrol exhibits significant anticancer activity in human breast and prostate cancer xenograft models, and that this is associated with increased apoptosis, decreased proliferation, and inhibition of angiogenesis. We demonstrate that targeting translation by silvestrol results in preferential inhibition of weakly initiating mRNAs. CONCLUSIONS/SIGNIFICANCE. Our results indicate that silvestrol is a potent anti-cancer compound in vivo that exerts its activity by affecting survival pathways as well as angiogenesis. We propose that silvestrol mediates its effects by preferentially inhibiting translation of malignancy-related mRNAs. Silvestrol appears to be well tolerated in animals.Canadian Institutes of Health Research (16512, Cancer Consortium Training Grant Award, CancerConsortium Training Grant Award); US Lymphoma Foundation Award; National Institute of Health (RO1 GM073855); National Crime Information Center (017099); Cole Foundation Awar

Keratin 8/18 Regulation of Cell Stiffness-Extracellular Matrix Interplay through Modulation of Rho-Mediated Actin Cytoskeleton Dynamics

Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18), hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells

ROCK involvement in K8/K18 IF modulation of cell stiffness.

<p>(A) Mean bead displacement curves of one data set containing for 400 FU beads attached to a monolayer of cells seeded on a 3 kPa gel, following addition of Y27632 (1 µM, 30 min). (B) The corresponding cell elastic constant k_c obtained in presence of vehicle (Ctrl) or Y27632. The k_c difference between H4ev and shK8b cell treated with Y27632 is not statistically significant (p = 0.26) (C) Mean bead displacement curves of one data set containing for 125 FU beads attached to a monolayer of cells seeded in a FN-coated dish, following addition of Y27632. (D) The corresponding cell elastic constant k_c obtained in absence (Ctrl) or presence of Y27632. The k_c difference between H4ev and shK8b cell treated with Y27632 is not statistically significant (p = 0.40). The dotted lines correspond to the numerical fits on Y27632-treated cells, while the solid lines correspond to the numerical fit on control cells data. Bars denote SE. *, p<0.05 relative to controls.</p

Substratum rigidity differentially affects H4ev and shK8b cell shapes.

<p>(A) Phase contrast images of cells one day after seeding on FN-gels of increasing rigidity or in FN-coated dishes, showing a more shK8b round cell shape under low FN-gel rigidity compared to the H4ev spread shape. For higher gel rigidity, both H4ev and shK8b cells show comparable spread shapes. (B) Measurements of H4ev and shK8b cell areas from the corresponding seeding conditions. N = 60. Bars denote SE. *, p<0.05 for H4ev versus shK8b.</p

H4ev and shK8b cell stiffness as function of bead FN-coating density.

<p>(A) Biomechanical model used to evaluate cell elastic and viscous parameters as function of the optical tweezers elastic constant and initial force. H4ev versus shK8b cells are seeded in FN-coated glass bottom dishes, which constitute a very high rigidity substrate (>3 GPa), and are allowed to form monolayers. Thereafter, beads exhibiting a FN coating of (B) 15, (C) 50, (D) 125 and (E) 400 fluorescent units (FU) are allowed to attach for 1 hr on the monolayers, and their displacements measured with the optical tweezers. Average displacement curves are generated from 40 independent bead measurements. The dotted curves present in each graph correspond to the numerical fit obtained from our mechanical model. The cell (F) elastic constant k_c and (G) viscosity constant γ_c are computed according to the model and the average is obtained from 3 separate experiments. Bars denote SE. *, p<0.05 for H4ev versus shK8b.</p

H4ev and shK8b cell stiffness as function of FN-gel rigidity.

<p>Mean bead displacement curves of one data set containing 40 independent bead (400 FU beads) measurements for cells plated on (A) 1.8 kPa gel and (B) 3 kPa gel for both H4ev and shK8b cells, along with the numerical fits (dotted line). (C) The corresponding 3- separate experiment averages of the computed elastic constant k_c, showing a differential stiffness increase from 1.8 kPa to 3 kPa in H4ev versus shK8b cells. *, p<0.05 relative to 1.8 kPa gels.</p

K8/K18 IF modulation of Rho-mediated actin fiber organization.

<p>(A) Confocal images of fibrillar actin at dorsal and ventral cell surfaces, following addition of Y27632 (1 µM, 1 h) on 3-day monolayers serum-starved overnight, showing that ROCK inhibition disrupts H4ev cell actin fiber organization at the basal and apical surface membranes to a greater extent in H4ev cells than in shK8b cells compare to untreated cells. (B) Western blottings of total Rho (A, B, C) and ROCK-1 showing increased Rho level in shK8b versus H4ev cells; Rho-GTP pull-down assay, showing a higher Rho activation in H4ev versus shK8b cells, despite a lower Rho (A, B, C) content. Confocal images of fibrillar actin at the ventral cell surface, showing cells expressing either a (C) RhoA-GFP or (D) a constitutively active mutant myc-RhoA-V17; RhoA-GFP expression induces the formation of dense actin fibers only in H4ev cells, while myc-RhoA-V17 induces the formation of comparable actin fibers in both H4ev and shK8b cells.</p

Characterization of the surfaceome of hematopoetic and leukemic stem cells to improve the development of novel therapies for leukemia.

Background Information: Hematopoietic stem cell transplantation (HSCT) has become a potent life-saving procedure for both adults and children with various hematological malignancies, including acute myeloid leukemia (AML). Umbilical cord blood (CB) appears as one of the most attractive sources of HSCs for transplantation, due to its immature immune system and widespread availability. Unfortunately, many patients are deprived from this therapeutic strategy as the low stem cell dose in CB units results in delayed engraftment and compromises transplantation outcome. Our collaborators from Guy Sauvageau’s group, have recently discovered a small-molecule, UM171, which stimulates the expansion of CB HSCs ex vivo. Interestingly, UM171 can also promote the in vitro maintenance of leukemic stem cells (LSCs), which are considered one of the major causes of therapeutic failure in AML. However, the unavailability of specific surface markers that can prospectively identify HSCs and LSCs is still a major hurdle for the optimization of CB grafts and the development of new targeted therapies. Purpose of the study: The aim of this collaborative research project is to identify new HSC and LSC surface markers using quantitative proteomic methods. These results will help better purify and characterize HSCs and LSCs in vitro, with the final purpose of helping patients with leukemic disorders either through the optimization of HSCT or the uncovering of new therapeutic targets. Methods: To discover novel and reliable HSC and LSC surface markers, we have optimized and adapted a cutting-edge chemoproteomic approach based on the labeling of cell surface proteins with cell-impermeable biotin reagents, their subsequent purification with avidin chromatography, and quantification using label-free quantitative proteomics with liquid chromatography-tandem mass spectrometry. Results: As a proof of concept experiment, we used this quantitative proteomic approach in the hematopoietic cell line OCI-AML5 in combination with UM171-induced cell expansion. We found that UM171 promotes the upregulation of more than 35 cell surface proteins. Among them, we identified the endothelial protein C receptor (EPCR) which we recently characterized as a novel surface marker for the HSC population derived from CB expanded with UM171. To optimize this method for the analysis of CB cells, we performed a OCI-AML5 cell titration and determined the minimal number of hematopoietic cells required. We then performed a surface proteomics analysis of cell populations from UM171-expanded CB units and sorted according to levels of CD34 and EPCR. Preliminary results reveal the enrichment in more than 100 surface proteins in the CD34+EPCR+ population, and several of these proteins are currently being tested as potential new markers for HSCs. Conclusion: Altogether, these results validate the surfaceome approach for the identification of novel HSC- and LSC-specific surface molecules using CB units and primary AML specimens, respectively

Define the surfaceome landscape of hematopoietic stem cells and pediatric leukemia specimens to improve the development of novel therapies for hematological diseases.

Background Information: Despite great advances in understanding the pathogenesis of various types of adult and childhood leukemias, there has been little progress in the development of new therapies and the 5-year survival rate remains low due to high incidence of relapse. Over the past decades, hematopoietic stem cell transplantation (HSCT) isolated from umbilical cord blood (CB) has evolved as a potent curative treatment intervention for patients with different type of blood disorders. Unfortunately, many patients are deprived of access to such therapies due to the low stem cell dose in CB units. The group of Dr. Guy Sauvageau and their collaborators have recently discovered a small-molecule, UM171, which promotes the ex vivo expansion of CB HSCs. However, the lack of reliable surface markers that can prospectively identify HSCs is still a major hurdle for the optimization of CB grafts. Purpose of the study: The aim of this collaborative research project is to identify novel and reliable HSC surface markers using a state-of-the-art surfaceomics approach. These results will help better purify and characterize HSCs in vitro, with the ultimate goal of developing novel strategies to achieve better ex vivo expansion of HSCs and generate optimized CB grafts. Methods: We have optimized and adapted a surface proteomic approach to simultaneously identify and quantify surface proteins in UM171-expanded CB cells. As a proof of concept experiment, we first used the hematopoietic cell line, OCI-AML5, which is responsive to UM171-induced cell expansion. To optimize this method for the analysis of CB cells, we performed a OCI-AML5 cell titration and determined the minimal number of hematopoietic cells required. Results: Using the recently discovered HSC marker, EPCR, we have performed a surfaceomics analysis of UM171-expanded CB HSCs and revealed the enrichment in more than 100 surface proteins in the CD34+EPCR+ population. In addition to well-known HSC markers (CD34, EPCR, CD133 and GPR56), we identified several surface proteins that may further subdivide the EPCR-positive population. To determine if these surface molecules are specific to HSC or other hematopoietic populations, their surface expression are being assessed by flow cytometry when antibodies are available. One such candidate could be GPA33, which appears to further subdivide the HSC population from CB cells. We are currently performing transplantation experiments in NSG mice to determine if GPA33 expression correlates with better engraftments. We are also producing unavailable antibodies against the other surface proteins to make possible their expression analysis via flow cytometry. Conclusion: Altogether, our published and preliminary data demonstrate the great potential of our surfaceomics method to identify novel and reliable HSC markers. The availability of the surfaceome landscape of HSCs will undeniably open new avenues for the optimization of HSCT. In addition, our potent surfaceomics approach will offer new opportunities for the development of antibody-based immunotherapies by uncovering targetable surface proteins from primary leukemia specimens