Mutations in the KRAS oncogene can mediate resistance to radiation. KRAS mutation-driven tumors have been reported to express cancer stem cell (CSC)-like features and may harbor metabolic liabilities through which CSC-associated radioresistance can be overcome. We established a radiation/drug screening approach that relies on the growth of 3D spheres under anchorage-independent and lipid-limiting culture conditions, which promote stemness and lipogenesis. In this format, we screened 32 KRAS mutation-enriched lung cancer models. As predicted from published data, CB-839, a glutaminase inhibitor, displayed the highest degree of radiosensitization in KRAS mutant models with LKB1 co-mutations. Radiosensitization by inhibition of stearoyl-CoA desaturase-1 (SCD1) displayed a similar genotype preference though the data also implicated KEAP1 co-mutation and SCD1 expression as potential predictors of radiosensitization. In an isogenic model, KRAS mutant cells were characterized by increased SCD1 expression and a higher ratio of monounsaturated fatty acids to saturated fatty acids. Accordingly, pharmacological inhibition or depletion of SCD1 radiosensitized isogenic KRAS mutant but not wild-type cells. The radiosensitizing effect was notably small, especially compared with several DNA repair inhibitors. As an alternative strategy to targeting monounsaturated fatty acid metabolism, adding polyunsaturated fatty acids phenocopied some aspects of SCD1 inhibition, suppressed tumor growth in vivo, and opposed the CSC-like phenotype of KRAS mutant cells. In conclusion, we report a 3D screening approach that recapitulates clinically relevant features of KRAS mutant tumors and can be leveraged for therapeutic targeting of metabolic vulnerabilities. Our data highlight pronounced intertumoral heterogeneity in radiation/drug responses and the complexity of underlying genomic dependencies. See related commentary by Buchsbaum, p. 840.

Peptide quantification from MIB column isolation of the active kinome in DMSO- versus dasatinib-treated HuCCT1, RBE, and SNU-1079 cells.

The mechanism of action of SHP099 inhibition

Cas9 and sgRNA expression plasmids.

Cell viability and Ln(IC50) for 885 cancer cell lines treated with dasatinib and saracatinib over nine doses.

RTK ligand expression in HNSCC cell lines

Characterization of the Osimertinib resistant H1975 cells.

Source and cell culture information for cell lines.

Effects of knockdown of Gab1 in SHP099-resistant HNSCC cell line

Weight profiles of xenograft models treated with SHP099

Western blot analysis of ErbB members

Supplementary Figure S1. IDHm ICC are insensitive to IDH inhibition. Supplementary Figure S2. Similarity matrix for drug response of BTC cell lines. Supplementary Figure S3. Hypersensitivity of ICC cells harboring endogenous IDH mutations to dasatinib. Supplementary Figure S4. Torin 1 inhibits growth of ICC cell lines. Supplementary Figure S5. Critical role for SRC in IDHm ICC cells. Supplementary Figure S6. Ectopic expression of mutant IDH has only a modest effect on dasatinib sensitivity.

HNSCC tumor cell lines are effective to SHP099 through dual PI3K and MEK inhibition

IC50 values for dasatinib in nM of nine ICC cell lines and the immortalized human cholangiocyte cell line MMNK-1.

Cell line information and SHP099 screen data

Adult liver malignancies, including intrahepatic cholangiocarcinoma and hepatocellular carcinoma, are the second leading cause of cancer-related deaths worldwide. Most individuals are treated with either combination chemotherapy or immunotherapy, respectively, without specific biomarkers for selection. Here using high-throughput screens, proteomics and in vitro resistance models, we identify the small molecule YC-1 as selectively active against a defined subset of cell lines derived from both liver cancer types. We demonstrate that selectivity is determined by expression of the liver-resident cytosolic sulfotransferase enzyme SULT1A1, which sulfonates YC-1. Sulfonation stimulates covalent binding of YC-1 to lysine residues in protein targets, enriching for RNA-binding factors. Computational analysis defined a wider group of structurally related SULT1A1-activated small molecules with distinct target profiles, which together constitute an untapped small-molecule class. These studies provide a foundation for preclinical development of these agents and point to the broader potential of exploiting SULT1A1 activity for selective targeting strategies.

Three- and five-day western blot analysis of SHP099-sensitive and - resistant HNSCC cell lines

Legends to supplementary table, data and figures

Median centered Ln(IC50) values for 17 BTC cell lines screened against 122 drugs.