a Average log2 fold changes (LFC) in abundance of mutant alleles of PDE3A following treatment with DNMDP as compared to treatment with DMSO. Wild-type (wt) alleles are marked with silent single-nucleotide polymorphisms (SNPs) (purple), whereas stop codons (red) result in complete loss of function until the last 59 amino acids. One standard deviation of increased survival is indicated with the dotted line. Resistance mutations with a survival score greater than one standard deviation from the mean and that are not predicted to disrupt PDE3A protein folding are colored to indicate localization to the active site (orange), homodimerization domain (green), or putative binding site of SLFN12 (dark blue). b Location of resistance mutations mapping to the PDE3A active site (orange), PDE3A homodimer interface (green), and putative SLFN12-binding site (dark blue). The backbone of the two PDE3A monomers are shown in light blue and white, with the mutations mapped onto the white monomer. The DNMDP is represented as space-filling and colored cyan. c Pulldown of wild-type or mutant PDE3A with a resin-conjugated DNMDP analog following ectopic expression in PDE3A-knockout A2058 cells. PDE3A proteins (marked with arrows) recovered in the pellet (P lanes) were visualized by western blotting using a PDE3A antibody. The presence of 10 μM trequinsin (T lanes) during pulldown competed away the binding of PDE3A to the resin, resulting in a negative pulldown. d Co-immunoprecipitation of ectopically expressed wild-type or mutant PDE3A with flag-tagged wild-type SLFN12 following ectopic expression in PDE3A-knockout HeLa cells. SLFN12 proteins, marked with arrows, that co-immunoprecipitated with PDE3A were detected by anti-FLAG western blotting. e Confirmation of deep mutation scanning results with selected PDE3A mutants in PDE3A-knockout A2058 cells. Seventy-two-hour Cell-Titer Glo assay. GFP, green fluorescent protein. f Dimer interface in the PDE3ACAT-Xtl structure. The labels for the residues are colored light and dark blue depending on which monomer they come from.