Figure 4. Discovery of CDK11 as the in cellulo target of the mis-characterized anti-cancer drug OTS964.

(A) A schematic of the strategy to use the highly mutagenic HCT116 cell line to isolate mutations that confer OTS964 resistance.

(B) Sanger sequencing validation of two heterozygous mutations in the CDK11B kinase domain.

(C) Constructs used to introduce the G579S mutation into CDK11B via CRISPR-mediated HDR. The yellow arrowhead indicates the site of Cas9 cleavage, the red bar indicates the G579S substitution, and the blue bars indicate silent mutations introduced to prevent re-cutting after HDR.

(D) Crystal violet staining of cancer cells transfected with the indicated constructs and then cultured in a lethal concentration of OTS964.

(E) 7-point dose-response curves of Rosa26, PBK-KO, and CDK11B^G579S clones grown in varying concentration of OTS964.

(F) Titration experiments reveal that OTS964 binds to CDK11B with a K_D of 40 nM.

(G) Pancreatic cancer cell line MiaPaca-2 was transduced with guides specific CDK11A, guides specific for CDK11B, or guides that harbored cut sites in both genes.

(H) A375 H2B-mCherry cells (left) or A375 H2B-mCherry cells that express CDK11B^G579S (right) were arrested at G1/S with a double-thymidine block and then were released into normal medium or medium containing OTS964. The percentage of mitotic cells in each population was scored every hour.

(I) Representative images of the experiments in (H), 9 hours after release from thymidine. Scale bar, 50 μm.