Psymberin, a marine-derived natural product, induces cancer cell growth arrest and protein translation inhibition

Abstract

Colorectal cancer (CRC) is the third most prevalent form of cancer in the United States and results in over 50,000 deaths per year. Treatments for metastatic CRC are limited, and therefore there is an unmet clinical need for more effective therapies. In our prior work, we coupled high-throughput chemical screens with patient-derived models of cancer to identify new potential therapeutic targets for CRC. However, this pipeline is limited by (1) the use of cell lines that do not appropriately recapitulate the tumor microenvironment, and (2) the use of patient-derived xenografts (PDXs), which are time-consuming and costly for validation of drug efficacy. To overcome these limitations, we have turned to patient-derived organoids. Organoids are increasingly being accepted as a "standard" preclinical model that recapitulates tumor microenvironment cross-talk in a rapid, cost-effective platform. In the present work, we employed a library of natural products, intermediates, and drug-like compounds for which full synthesis has been demonstrated. Using this compound library, we performed a high-throughput screen on multiple low-passage cancer cell lines to identify potential treatments. The top candidate, psymberin, was further validated, with a focus on CRC cell lines and organoids. Mechanistic and genomics analyses pinpointed protein translation inhibition as a mechanism of action of psymberin. These findings suggest the potential of psymberin as a novel therapy for the treatment of CRC.

Keywords: high-throughput screening; patient-derived models of cancer; patient-derived organoids; precision medicine; protein translation; psymberin.

FIGURE 1

Identification of psymberin as a potential anti-cancer therapeutic agent for colorectal cancer (CRC). (A) Tumor samples or low-passage cell lines from patients are used to establish organoid cultures ex vivo. These organoids are then used in drug screens. (B) A high-throughput screen with 400 compounds in six cell lines. Blue indicates a negative average percent killing (net growth) and red indicates a high average percent killing. Compounds that were not tested for a cell line are shown in gray. (C) The top ten hits from the high throughput screen in the same six cell lines. Some compounds were synthesized in multiple batches and were therefore tested more than once. (D) Dose-response comparison between oxaliplatin and psymberin in the same CRC cell line (left) and comparison of psymberin IC-50 values across different CRC cell lines (right).

FIGURE 2

RNA-Seq analysis of psymberin treatment in colorectal cancer (CRC) cell lines. (A) Upset plot showing overlap of up- and down-regulated genes after psymberin treatment between two CRC cell lines: CRC119 and CRC16-159. Red indicates the number corresponding to upregulated genes consistent between both lines; blue indicates the number of genes consistently downregulated in both lines. (B) Same as 3A with coding genes only. (C) Most significant positively enriched pathways after psymberin treatment, determined using gene set enrichment analysis. (D) Most significant negatively enriched pathways after psymberin treatment, determined using gene set enrichment analysis. *Corresponds to Reactome, **Kegg, and ***Hallmark.

FIGURE 3

Psymberin induces protein synthesis inhibition and p38 activation. (A) Protein synthesis assay in CRC119 cells that were treated with 20 nM psymberin for one and 6 h (top). Quantification of fluorescence in protein synthesis assay (bottom) *p < 0.05. (B) Western blot for phospho-p38 in CRC119 cells treated with 20 nM psymberin at different time points. Total p38 and GAPDH are included as loading controls. (C) Western blot for cleaved PARP in CRC119 cells treated with 20 nM psymberin at different time points. GAPDH is included as a loading control. (D) Annexin V staining for protein translation in CRC119 and CRC16-159 at 0 and 24 h after treatment with psymberin.

FIGURE 4

Psymberin has IC-50 values in the nanomolar level across multiple colorectal cancer (CRC) patient-derived organoids. Dose response curves are shown for six different CRC patient-derived organoids. Each experimental repeat is depicted in a different curve with different IC-50 values listed on the side of each curve. Images beside each graph show organoids from each line treated with 320 pM (left) and 1 μM (right) of psymberin.

FIGURE 5

The entire structure of psymberin is important for its activity. (A) Structural comparison of psymberin and two of its analogs, Psy-064 and Psy-076. (B) Dose response curves for psymberin, Psy-064, and Psy-076 in CRC404 organoids. Images below each graph show organoids from each line treated with 1 μM (top) and 0.0002 μM (bottom) of that compound.