Immunoblot analyses of CrkL phosphorylation For cell line experiments, Ba/F3 cells expressing BCR-ABL, BCR-ABLE255V, or BCRABLT315I have been cultured four h in full media alone or with DCC-2036 or imatinib as described . For principal cell experiments, following informed consent, Nutlin-3 peripheral blood mononuclear cells from a newly diagnosed CML patient and from an accelerated phase patient harboring BCR-ABLT315I have been cultured overnight in IMDM medium containing 20% BIT alone or with DCC-2036 , imatinib , nilotinib , or dasatinib . For all experiments, cells were lysed in boiling SDS-PAGE loading buffer supplemented with 0.1 mmol/L AEBSF and 0.1 mmol/L Na3VO4, subjected to SDS-PAGE, and immunoblotted with antibodies towards phosphorylated or total CrkL . Hematopoietic colony formation assays To assess granulocyte/macrophage colony formation , mononuclear cells from bone marrow of the newly diagnosed CML patient, an accelerated phase patient harboring BCR-ABLT315I, or possibly a wholesome donor have been obtained following informed consent and plated alone or with DCC-2036 or imatinib in triplicate in IMDM:methylcellulose as described . Success are reported as percentage of colonies relative to untreated. Cell-based resistance screens For DCC-2036 screens beginning from Ba/F3 cells expressing native BCR-ABL, cells have been treated overnight with N-ethyl-N-nitrosourea and resuspended in total media supplemented with DCC-2036 as described .
DCC-2036 was also evaluated in dual-combinations with imatinib , nilotinib , or dasatinib . Wells exhibiting jak2 inhibitor selleck outgrowth were expanded, sequenced, and analyzed for mutations as described . Very similar experiments were performed commencing from Ba/F3 BCR-ABLT315I cells treated with DCC-2036 , and from a pooled mix of equal cell numbers of all Ba/F3 BCR-ABL cell lines handled with a cocktail of ABL kinase inhibitors / nilotinib / dasatinib ).
Final results and Discussion We established that DCC-2036 directly inhibits the catalytic action of ABL and ABLT315I by evaluating kinase autophosphorylation exercise. Even though the two imatinib and DCC-2036 attenuated activity of ABL, only DCC-2036 blocked ABLT315I tyrosine autophosphorylation . Unlike imatinib, nilotinib, and dasatinib, the binding mode of DCC-2036 to ABL or ABLT315I doesn’t require a hydrogen bond for the side chain hydroxyl of native T315 and avoids a steric clash with mutated I315. Upon binding, DCC-2036 induces and stabilizes a DFG-out, catalytically inactive conformation in the kinase domain, precluding phosphorylation of activation loop residue Y393, a vital event preceding complete catalytic activation of ABL kinase1 Cellular assays demonstrated that DCC-2036 selectively inhibits most clinically appropriate imatinib-resistant mutants . DCC-2036 inhibited growth of Ba/F3 cells expressing BCR-ABL with ~16-fold greater potency than imatinib and, of essential value, cells expressing BCR-ABLT315I .