No abstract

Imatinib mesylate (IM), a potent inhibitor of the BCR/ABL tyrosine kinase, has become standard first-line therapy for patients with chronic myeloid leukemia (CML), but the frequency of resistance increases in advancing stages of disease. Elimination of BCR/ABL-dependent intracellular signals triggers apoptosis, but it is unclear whether this activates additional cell survival and/or death pathways. We have shown here that IM induces autophagy in CML blast crisis cell lines, CML primary cells, and p210 BCR/ABL -expressing myeloid precursor cells. IM-induced autophagy did not involve c-Abl or Bcl-2 activity but was associated with ER stress and was suppressed by depletion of intracellular Ca 2+ , suggesting it is mechanistically nonoverlapping with IM-induced apoptosis. We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of essential autophagy genes enhanced cell death induced by IM in cell lines and primary CML cells. Critically, the combination of a tyrosine kinase inhibitor (TKI), i.e., IM, nilotinib, or dasatinib, with inhibitors of autophagy resulted in near complete elimination of phenotypically and functionally defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML.

Bcr-Abl is a dysregulated tyrosine kinase whose mechanism of activation is unclear. Here, we demonstrate that, like c-Abl, Bcr-Abl is negatively regulated through its SH3 domain. Kinase activity, transformation, and leukemogenesis by Bcr-Abl are greatly impaired by mutations of the Bcr coiled-coil domain that disrupt oligomerization, but restored by an SH3 point mutation that blocks ligand binding or a complementary mutation at the intramolecular SH3 binding site defined in c-Abl. Phosphorylation of tyrosines in the activation loop of the catalytic domain and the linker between the SH2 and catalytic domains (SH2-CD linker) is dependent on oligomerization and required for leukemogenesis. These results suggest that Bcr-Abl has a monomeric, unphosphorylated state with the SH3 domain engaged intramolecularly to Pro1124 in the SH2-CD linker, the form that is sensitive to the inhibitor imatinib (STI-571). The sole function of the coiled-coil domain is to disrupt the autoinhibited conformation through oligomerization and intermolecular autophosphorylation.