Constraining regulatory domain dynamics of the Src kinase Fgr increases ATP-site inhibitor sensitivity and impairs bone marrow engraftment.

Acute myeloid leukemia is often associated with constitutive activation of the Src-family kinases, Hck, Lyn, and Fgr. Their modular SH3 and SH2 domains regulate kinase activity and signal transduction. Here, we show that regulatory domain dynamics critically influence both inhibitor sensitivity and leukemogenic signaling. We modified the Fgr SH2-kinase linker to enhance intramolecular SH3 engagement, shifting the conformational ensemble to the closed state. This shift increased the Km for ATP and enhanced the potency of ATP-site inhibitors in vitro. Human myeloid cells expressing these constrained Fgr variants exhibited heightened sensitivity to ATP-site inhibitors in terms of growth arrest. These cells also demonstrated impaired bone marrow engraftment in vivo, suggesting a key role for Fgr dynamics and SH3-dependent signaling in leukemia cell survival within this niche. Small molecules that similarly restrict Src-family kinase regulatory domain dynamics may provide a new therapeutic approach to AML and other cancers linked to these kinases.