Harnessing Multivalency and Oligomerization for the Selective Inhibition of ETO2 and Related MYND-Containing Proteins

ETO proteins are cofactors involved in gene regulation and have been implicated in several hematological disorders. Recent work by the Dean lab showed that ETO2 contributes to NuRD recruitment at the -globin locus; however, the molecular determinants governing this interaction remain unclear.1 In this work, we investigated the interaction between ETO2 and NuRD and found that ETO2-NHR4 binds weakly to the four individual PPPL motifs in GATAD2A-NuRD, but exhibits increased binding affinity through multivalent engagement of multiple motifs. Furthermore, inclusion of the NHR3 coiled-coil domain further enhances binding through weak self-association. Leveraging these principles, we developed a high-affinity bivalent peptide inhibitor incorporating a similar poly-proline motif from N-CoR, enhancing the affinity for ETO2-NHR3-4 nearly 40-fold. Enforced expression of this bivalent peptide stimulated fetal hemoglobin expression and induced differentiation in K562 leukemia cells. In parallel, PKA(RII)α, which binds directly to the NHR3 domain, reduced the affinity of ETO2-NHR3-4 for multivalent peptides by approximately 30-fold and produced similar biological effects. These findings establish that oligomerization of ETO2-NHR3 and multivalent binding mediated by ETO2-NHR4 both play a vital role in the association of ETO2 with co-regulatory complexes. In addition to oligomerization and multivalency, we identified that linker composition impacts binding and promotes selective binding among MYND-containing proteins. ETO2 preferentially binds constructs with shorter linkers, while ZMYND8 favors peptides with longer linker lengths. Application of these principles enabled the development of a high-affinity, multivalent, selective binder of ZMYND8, demonstrating that linker configuration can be exploited to promote selectivity. Together, these findings advance the understanding of the use of oligomerization and multivalency in co-regulatory protein recruitment mediated by MYND-containing proteins such as ETO2 and ZMYND8 and provide a foundation for therapeutic strategies targeting ETO2 and MYND-containing protein-mediated transcriptional repression.