Abstract 54: Cryo-EM structure of the PDE3A-SLFN12 complex reveals requirements for the activation of SLFN12 RNase and DNMDP-induced cancer cell killing

We have identified a novel mechanism of selective cancer cell-killing, whereby small molecules such as DNMDP induce complex formation between phosphodiesterase 3A (PDE3A) and a poorly characterized protein, SLFN12, that leads to downstream cell death (de Waal et al., Nat Chem Biol 2016; Lewis et al., ACS Med Chem Letters, 2019; Wu et al., J Bio Chem 2020). This process therefore relies on the presence and interaction of these two proteins, rather than a pre-existing dependency, as is the mechanism of many currently available cancer therapies. While complex formation is required for cancer cell killing, the molecular basis for complex induction and the activity of the complex that leads to cell death have not been described. In this study, we employed cryogenic electron microscopy (cryo-EM) to deduce the high-resolution structure of the DNMDP-induced PDE3A-SLFN12 complex. The solved structure includes DNMDP-bound PDE3A at 2.97 Å, as well as the structure of the full length SLFN12 at 2.76 Å. We found that the entire complex consists of a 2:2 hetero-tetramer with the C-terminal alpha-helix of SLFN12 reaching into the DNMDP-occupied substrate binding pocket of PDE3A and contacting both DNMDP and active site residues of PDE3A. The binding of DNMDP to PDE3A does not induce structural changes, but rather creates an adhesive surface at the entrance of the substrate binding pocket, which allows for stable binding of SLFN12. We have named DNMDP and similar PDE3A-SLFN12 complex inducing compounds “velcrins”, after the adhesive fabric. Site-directed mutagenesis confirmed that residues on the SLFN12 C-terminal helix are required for DNMDP-induced cell death. Based on homology to the SLFN13, we predicted that SLFN12 would also encode an RNase. Indeed, purified SLFN12 protein cleaved ribosomal RNA in vitro and mutation of the putative catalytic residues abolished this activity. Importantly, catalytically dead SLFN12 mutants failed to mediate DNMDP-induced cell killing while retaining the ability to complex with PDE3A. Consistent with the requirement of SLFN12 RNase activity for DNMDP-induced cell death, overexpression of wild-type SLFN12 alone was cytotoxic, whereas overexpression of catalytically dead SLFN12 was not. The RNase activity of SLFN12 in vitro was stimulated in the presence of DNMDP and PDE3A, suggesting that the mechanism of DNMDP-induced cell killing involves activation of the SLFN12 RNase. Intriguingly, SLFN12 homodimer interface mutants were no longer cyt