Article Figures & Data
Figures
- Figure 1.
Clinical kinase inhibitors for HCC share common structural features that enable conformation specific binding. A, Sorafenib and regorafenib are both approved for first-line and second-line treatment of HCC; their chemical structures differ by a single fluorine. Cabozantinib and lenvatinib are also promising therapies for HCC. All four compounds share several conserved structural motifs: the hinge-binding element functions as an adenosine mimic, the linker and cap extend deep into the kinase active site pocket. B, Top, Phosphorylated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog within an active state DFG (aspartate–phenylalanine–glycine)-IN configuration (PDB ID: 1IR3), where the ATP analog is able to interact with the DFG loop, and thus amenable to both substrate recognition and phosphorylation (bottom). C, Top, binding of sorafenib to BRAF (PDB ID: 1UWH) forces the kinase to adopt the DFG-OUT conformation. In this state, the BRAF activation-segment (A-seg; bottom) adopts an inactive conformation. The DFG-OUT form of binding is referred to as type II inhibition.
- Figure 2.
The type II inhibitor AD80 is highly active across multiple in vitro models of HCC. A–F, HCC and hepatocyte cell lines incubated with 0.3 nmol/L to 20 μmol/L of AD80, sorafenib, and regorafenib in 0.1% DMSO (n ≥ 3 technical). G, Calculated GI50 values of drugs from A to F on cell lines (mean ± SD in μmol/L). H, Clonogenic crystal violet assay of THLE5B, immortalized hepatocyte cell line, and HUH7, treated under the indicated conditions for 14 days. Each condition was tested in triplicate. I, Therapeutic window calculated on the basis of normalization of GI50 values from normal (THLE5B) and transformed (HUH7) lines for the indicated compounds.
- Figure 3.
AD80 significantly improves survival in a preclinical HCC model through enhanced inhibition of the ERK/MAPK pathway. A, Mouse survival in a HUH7 xenograft model. B, AD80-treated animals show a tumor growth rate that is half of vehicle-treated animals, and significantly less than sorafenib-treated animals. C, Western blot analysis of individual tumors harvested from animals at the end of the survival experiment shown in D. Note that mice that survived longest in D displayed relatively weak phospho-ERK1/2 signal compared with mice that died early. Antibodies used were as follows: S6 Ribosomal Protein(S6RP; 5G10), phospho-S6 Ribosomal Protein(S6RP; Ser235/236, Phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204), p44/42 MAPK (Erk1/2; 137F5). Blots were run in duplicate; representative data are shown.
- Figure 4.
AD80 induces an increased metabolism and reduced oncogenic signaling gene signature compared with sorafenib in HUH7 cells. The AD80-induced gene signature significantly correlates to greater overall survival and lower AFP levels based on TCGA-LIHC dataset. A, Heatmap of metabolism and MAPK-associated genes significantly upregulated or downregulated across the three groups within the indicated cell lines. Color key denotes the Z-score (number of SDs from the mean) of the sample and gene row. B, GSEA based on HUH7 data against the Hallmark Gene Sets, where normalized enrichment scores are shown as bars and FDR < 0.05 is marked by an asterisk. For A and B, three biological replicates per treatment near the IC50 dose (sorafenib 5 μmol/L, AD80 50 nmol/L, DMSO 0.1%) on HUH7 or THLE5B cell lines as indicated, over a course of 24 hours, were completed. C, Kaplan–Meier survival comparison of TCGA-LIHC patient samples, separated by patients with (+) or without (−) the genes upregulated/downregulated by AD80 (Supplementary Table S3). Log-rank (Mantel–Cox) testing used to determine significance. Summary of number of patients at risk every 2.5 years, separated on the basis of AD80 signature shown below the Kaplan–Meier. D, Box and whisker plot of TCGA-LIHC AD80 signature.
- Figure 5.
Activity-based protein profiling by mass spectrometry identifies kinases strongly inhibited by AD80 in the HUH7 cell line. % inhibition (y-axis) corresponds to the difference in kinase labeling with ATP-desthiobiotin in AD80-treated samples compared with untreated controls for each kinase shown along the x-axis. AD80-treated and -untreated samples were analyzed in duplicate and quadruplicate, respectively. See Supplementary Table S2 for raw profiling data.
- Figure 6.
AD80 is isoform-biased for p38γ and p38δ, over p38α and p38β, with binding dependent on the gatekeeper residue. A, Chemical structure of AD80 and related compounds. B, Cell viability assay comparing activity of each compound on the HUH7 cell line. Technical replicates n = 6. C, Hierarchical clustering of previously generated in vitro–inhibitory profiles (28) of the most different kinases between the related compounds. D and E, Melting temperature binding assay using a 1:1 ratio of drug to protein (i.e., 5 μmol/L drug to 5 μmol/L protein). Each point is the difference in mean melting temperature (technical replicates: n = 6) as determined by differential scanning fluorimetry. In E, the following mutations at the gatekeeper residue were tested: p38α/MAPK14 T106M, p38β/MAPK11 T106M, p38γ/MAPK12 M109T, p38δ/MAPK13 M107T.
- Figure 7.
Low mRNA expression of p38γ and p38δ correlates with significantly better overall survival for individuals with liver cancer. A–D, Kaplan–Meier overall survival curves for individuals with low (blue) and high (red) p38 isoform expression based on liver cancer TCGA data compiled from the Protein Atlas. Expression cutoffs between high versus low were 0.7 Fragments Per Kilobase of transcript per Million mapped reads (FPKM; MAPK12), 0.2 FPKM (MAPK13), 9.6 FPKM (MAPK14), and 2.7 FPKM (MAPK11). In all panels, P values were determined using log-rank (Mantel–Cox) testing.
Additional Files
Supplementary Data
- Supplementary Data Tables 1-3 - Supplemental Table 1 shows RNA-seq signature genes. Supplementary Table 2 shows mass spectrometry profiling data for AD80 in HUH7. Supplementary Table 3 shows in vitro profiling data on compounds.
- Supplementary Data Figures 1-7 - Supplementary Data Figures 1 shows PK data on AD80. Supplementary Data Figure 2 shows PCA analysis. Supplementary Data Figure 3 shows Gene Set Enrichment of HUH7 cells in response to AD80 and sorafenib. Supplementary Data Figure 4 shows western blot data. Supplementary Data Figure 5 shows representative purification gels of p38 isoforms used in the study. Supplementary Data Figure 6 shows a p38 sequence alignment. Supplementary Data Figure 7 shows rescue data.
- Supplementary Synthetic Methods - Supplemental Synthetic Methods
Volume 18, Issue 9
