CRISPR-Cas9 Genome-Wide Knockout Screen Identifies Mechanism of Selective Activity of Dehydrofalcarinol in Mesenchymal Stem-like Triple-Negative Breast Cancer Cells

Abstract

There are no targeted therapies available for triplenegative breast cancers (TNBCs) in part because they represent a heterogeneous group of tumors with diverse oncogenic drivers. Our goal is to identify targeted therapies for subtypes of these cancers using a mechanism-blind screen of natural product extract libraries. An extract from Desmanthodium guatemalense was 4-fold more potent for cytotoxicity against MDA-MB-231 cells, which represent the mesenchymal stem-like (MSL) subtype, as compared to cells of other TNBC subtypes. Bioassay-guided fractionation led to the isolation of six polyacetylenes, and subsequent investigations of plant sources known to produce polyacetylenes yielded six additional structurally related compounds. A subset of these compounds retained selective cytotoxic effects in MSL subtype cells. Studies suggest that these selective effects do not appear to be due to PPARγ agonist activities that have previously been reported for polyacetylenes. A CRISPR-Cas9-mediated gene knockout screen was employed to identify the mechanism of selective cytotoxic activity of the most potent and selective compound, dehydrofalcarinol (1a). This genomic screen identified HSD17B11, the gene encoding the enzyme 17β-hydroxysteroid dehydrogenase type 11, as a mediator of the selective cytotoxic effects of 1a in MDA-MB-231 cells that express high levels of this protein. The Project Achilles cancer dependency database further identified a subset of Ewing sarcoma cell lines as highly dependent on HSD17B11 expression, and it was found these were also highly sensitive to 1a. This report demonstrates the value of CRISPR-Cas9 genome-wide screens to identify the mechanisms underlying the selective activities of natural products.

SDR enzymes oxidize specific lipidic alkynylcarbinols into cytotoxic protein-reactive species

“…The copyright holder for this this version posted September 15, 2021. ; https://doi.org/10.1101/2021.09.15.460423 doi: bioRxiv preprint p.6/55 synthetic derivatives tested. In addition, HSD17B11 has been recently identified as mediating, through an unknown mechanism, the cytotoxic effect of dehydrofalcarinol, a polyacetylenic compound with a terminal butadiynylalkenylcarbinol motif isolated from several plants of the Asteraceae family (21).…”

Altertoxin II, a Highly Effective and Specific Compound against Ewing Sarcoma

“…In the future, we plan to conduct additional binding studies with radiolabeled ATXII, which will be used to identify its direct molecular target(s). Additionally, a CRISPR-Cas9-mediated gene knockout screen will be employed to further define the mechanism of ATXII-induced DNA damage in ES cells [ 55 ]. Lastly, the development of an ATXII-resistant ES cell line, followed by whole-exome and RNA sequencing, would be useful in identifying both genetic and epigenetic mechanisms of ATXII resistance, and may provide further insight into its mechanisms of action.…”

The Natural Products and Extracts: Anti‐Triple‐Negative Breast Cancer in Vitro

“…It is shown that HSD17B11, the gene encoding the enzyme 17β-hydroxysteroid dehydrogenase type 11, was a mediator of the selective cytotoxic effects of 26a in MDA-MB-231 cells. [57] There are other natural products from terrestrial with anti-TNBC activities (Table 1) (Figure 10).…”

“…A CRISPR‐Cas9‐mediated gene knockout screen was employed to identify the mechanism of selective cytotoxic activity. It is shown that HSD17B11, the gene encoding the enzyme 17β‐hydroxysteroid dehydrogenase type 11, was a mediator of the selective cytotoxic effects of 26a in MDA‐MB‐231 cells [57] …”