Targeting the SH2-Kinase Interface in Bcr-Abl Inhibits Leukemogenesis

Abstract

SUMMARY Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface

“…1C). Expression of this tandem monobody construct suppressed Bcr-Abl-dependent oncogenic transformation of mouse bone marrow cells by inhibiting Bcr-Abl kinase activity and induced apoptosis in human cells isolated from CML patients (9). These results supported the notion that the SH2-kinase interface is a potentially druggable site.…”

“…1B). Consequently, a mutation that disrupts this interface, I164E, inhibits enzyme activity and Bcr-Abl-mediated oncogenesis (8,9). These results suggest that the SH2-kinase interface is a potentially "druggable" site for allosteric inhibition of the kinase activity.…”

“…Because monobodies are stable and, unlike antibody fragments, do not contain a disulfide bond, they are well suited as genetically encoded inhibitors of an intracellular target. We successfully generated a monobody termed "7c12" directed to the kinase-binding surface of the Abl SH2 domain (9). Because the 7c12 monobody had only moderate affinity and hence moderate biological effects in vitro and in cells, we needed to fuse it with another monobody, termed HA4, binding to a different surface of the SH2 domain, namely the binding site for phospho-Tyr-containing ligands (13).…”

“…In our previous work, to determine the feasibility of disrupting the SH2-kinase interface in trans, we utilized synthetic binding proteins, termed "monobodies," to target this interface (9). Monobodies are binding proteins developed from combinatorial libraries constructed on the molecular scaffold of a fibronectin type III domain (10 -12).…”

Two-state dynamics of the SH3–SH2 tandem of Abl kinase and the allosteric role of the N-cap

“…For Abl in particular, the significance of this mechanism is underscored by the fact that mutations that impair the correct assembly of the autoinhibited complex, either in the SH3-SH2 tandem or in the domain-domain linkers, confer CML cells with resistance against inhibitory drugs designed to target the catalytic domain of the Bcr-Abl oncogene (14). This outcome has prompted considerable interest in the mechanisms of allosteric regulation of Abl and other tyrosine kinases and in the development of compounds designed to interfere with these mechanisms (15)(16)(17)(18)(19).…”