Journal: Science Advances

Article Title: Cell-penetrating Alphabody protein scaffolds for intracellular drug targeting

doi: 10.1126/sciadv.abe1682

Figure Lengend Snippet: ( A ) Addition of an N- and C-terminal arginine-proline ([RP] 7 ) tag allows bilayer phospholipid crossing of CMPX-321A, the CPAB-formatted successor of CMPX-152A. ( B ) Western blot of the rest fraction (RF) and cytosolic fraction (CF) of NCI-H929 cells after 2 hours of incubation with 2 μM of the CPAB CMPX-321A. ( C ) Overlay of immunofluorescence experiments confirming the cell-penetrating capacity of CMPX-321A (green) in HeLa cells, after 30 min of incubation with 1 μM Alphabody. Nuclei are stained with Ruby stain (red). ( D ) RNA expression data ( , ) show that MCL-1 is substantially more expressed in MM cells compared to other cancer cell types, indicating that this cancer cell type might be MCL-1 dependent. ( E ) The cell killing potential of CMPX-321A was evaluated via a panel of 33 MM cell lines. Thirteen MM cell lines were shown to be very sensitive toward MCL-1 inhibition (IC 50 < 1 μM), 10 cell lines were moderately sensitive (IC 50 = 1 to 2 μM), and 6 cell lines were not sensitive to unresponsive (IC 50 > 2 μM). ( F ) The cell killing potency of CMPX-321A cannot be predicted on the basis of the classical molecular signature of MM cell lines (CCDN1, MAF, MMSET, and others). FPKM, fragments per kilobase million.

Article Snippet: A survey of RNA expression data from the Broad Institute Cancer Cell Line Encyclopedia (Entrez ID 4170, accessed 29 March 2020) ( , ) shows that the expression levels of MCL-1 in MM cells is significantly higher than in other cancer types ( ).

Techniques: Western Blot, Incubation, Immunofluorescence, Staining, RNA Expression, Inhibition

Journal: iScience

Article Title: Profiling of diverse tumor types establishes the broad utility of VHL-based ProTaCs and triages candidate ubiquitin ligases

doi: 10.1016/j.isci.2022.103985

Figure Lengend Snippet: Correlation of PROTAC activity with CRBN and VHL RNA expression, DNA copy number, and protein level (A and B) DC50 values from the cell line panel for each compound were used to group the cell lines into the bottom (low) and top (high) quartiles. Low and high quartiles were plotted against ligase mRNA expression or copy number, p values from unpaired two-samples, two-sided Wilcoxon rank sum tests of the groups were calculated. (A) dBET1 activity correlates significantly with CRBN copy number, p = 0.00058, and mRNA expression, p = 0.0048. Cell lines with non-synonymous mutations of CRBN were marked in red. (B) MZ1 activity correlates with VHL RNA expression, p = 0.028 but not VHL copy number, p = 0.059. Cell lines with non-synonymous mutations of VHL were marked in red. (C) Dose-response curves from representative kidney-derived cancer cell lines are shown; 786-O is devoid of both VHL and CRBN activity, 769P is lacking VHL activity. Dose titration curves are derived from n = 2 independent experiments, error bars represent standard error of the mean (SEM). (D) Lysates from untreated cells were separated by capillary electrophoresis, VHL and CRBN proteins were immune-detected. Each of the five kidney-derived cancer cell lines is lacking VHL protein, all of the cell lines express appreciable CRBN protein. (E) Dose-response curves from two representative lung cancer cell lines, H23 lacks dBET1-CRBN-associated activity. Dose titration curves are derived from n = 2 independent experiments, error bars represent standard error of the mean (SEM). (F) Lung-derived cancer cell lines with low CRBN activity have low or no CRBN protein, all lung cancer cell lines express VHL protein.

Article Snippet: For this analysis, we retrieved RNA-Seq expression and DNA alteration data from available OmicSoft TCGA data sets and graphed these by TCGA-designated tumor type ( B).

Techniques: Activity Assay, RNA Expression, Expressing, Derivative Assay, Titration, Electrophoresis

Journal: iScience

Article Title: Profiling of diverse tumor types establishes the broad utility of VHL-based ProTaCs and triages candidate ubiquitin ligases

doi: 10.1016/j.isci.2022.103985

Figure Lengend Snippet: Comparison of genomic features for seven PROTAC Ub-ligases (A) Boxplot showing the distribution of log transformed fold change of the expression of seven Ub-ligases in cancer tissue compared to its paired non-cancer control tissue. Only cancer types with at least 50 non-cancer control tissue samples were included. (B) DNA alternation landscape of seven Ub-ligases across cancer types in TCGA. Bar graph showing percentage of samples harboring each Ub-ligase mutations across tumor types, the number of samples altered are labeled in the right side of each bar. Different mutation types are color labeled, with red representing amplification, blue representing homozygous deletion, green representing non-synonymous mutations, and gray representing a mixture of the above type of mutations. (C) Genome-scale CRISPR-Cas9 essentiality screen results for genes in across different cancer cell lines performed by the Broad Institute were characterized by dependency score (CERES) to reflect the functional importance of genes in certain cancer types. Boxplots summarized the distribution of CERES score of each ligase receptor in cell lines of representative tumor types. A lower score means that a gene is more likely to be essential for the cancer cell line survival and proliferation. A score of −1 corresponds to the median of all common essential genes, used as a cutoff indicator here.

Article Snippet: For this analysis, we retrieved RNA-Seq expression and DNA alteration data from available OmicSoft TCGA data sets and graphed these by TCGA-designated tumor type ( B).

Techniques: Comparison, Transformation Assay, Expressing, Control, Labeling, Mutagenesis, Amplification, CRISPR, Functional Assay