Dasatinib (BMS-354825) Has Increased Activity Against Bcr-Abl Compared to Imatinib in Primary CML Cells In Vitro, but Does Not Eradicate Quiescent CML Stem Cells.

Abstract

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder of the haemopoietic stem cell. It results from acquisition of the Philadelphia chromosome and expression of the oncogenic fusion protein Bcr-Abl. Imatinib mesylate (IM), a rationally designed tyrosine kinase inhibitor of Bcr-Abl, competitively inhibits ATP binding for which conformation of Bcr-Abl is critical. IM induces a complete cytogenetic response in the majority of CML patients in chronic phase, but nearly all patients have detectable disease at the molecular level by quantitative RT-PCR and, therefore, are unlikely to be cured. Dasatinib is a novel, oral, multi-targeted kinase inhibitor that targets Bcr-Abl and Src kinases, and is currently in Phase 2 clinical trials in CML. In vitro, dasatinib has improved (325 fold greater) potency against wild-type Bcr-Abl expressing cells and is capable of binding Bcr-Abl conformations resistant to IM1,2 and it is proposed that dasatinib will eradicate CML in the majority of patients regardless of mutation status (except T315I). To test this, CD34+ primary CML cells were cultured for 6 days in serum free medium supplemented with 5 growth factors. CFSE was used to track cell division. Conditions included: (1) no drug control, (2) continuous IM (5μM; ~IC90 dose), (3) continuous dasatinib (150nM; ~IC90 dose) (4) continuous IM/dasatinib, (5) IM (72hr) then dasatinib (72hr), (6) dasatinib (72hr) then IM (72hr). Crkl phosphorylation status was evaluated by intracellular flow cytometry in CD34+ and CD34+CD38− primary CML cells at 16 and 72 hours as a marker of kinase activity. Both CD34+ and CD34+CD38− cells showed only single copy Bcr-Abl by FISH, but expressed significantly higher Bcr-Abl transcript levels by RT-PCR compared to total mononuclear cells (P=0.031). There was a significant reduction in total viable cells in all treatment arms versus the no drug control (P=0.003). There was accumulation of undivided CFSEmax CD34+ CML cells in all treatments arms relative to the no drug control (P=0.009). There were no significant differences in undivided CFSEmax CD34+ CML cells remaining after 6 days between individual arms, but, collectively, the arms containing IM had significantly greater accumulation of these cells compared to the non-IM containing arms (P=0.045). Total CD34+ cells showed dephosphorylation of Crkl at 16 hours after treatment with IM or dasatinib. However, after 72 hours, the remaining viable CD34+ Bcr-Abl+ cells showed no dephosphorylation of Crkl in response to IM, consistent with enrichment of a resistant population, whereas the dasatinib-treated cells remained dephosphorylated (P=0.01). In the CD34+38− sub-population, there was no Crkl dephosphorylation at 16 or 72 hours with IM, however, dasatinib induced 43% and 50% dephosphorylation at 16 and 72 hours respectively (P=0.009 and P=0.001). Kinase domain mutations were not detected in either the IM or dasatinib-resistant primitive CML cells. These results demonstrate dasatinib is more effective than IM within the stem cell compartment, however, the most primitive quiescent CML cells remain insensitive to both drugs, questioning the relevance of Bcr-Abl as a therapeutic target in these cells.

Chronic Myeloid Leukemia: Diagnosis and Treatment

“…148 Emerging data suggest that resistance of quiescent cells to imatinib is indeed a Bcr-Abl-dependent phenomenon. 149 Other potential mechanisms implicated in quiescent CML progenitors include expression of drug transport proteins such as PgP and Abcg2, atypical kinase domain mutations, 150 and BCR-ABL overexpression. 151 Monitoring of Imatinib Therapy and Minimal Residual Disease.…”

“…Despite the impressive results achieved with dasatinib, it may not eradicate all quiescent leukemic stem cells, although it may be more effective at this level than imatinib. 149 Another dual Abl/Src kinase inhibitor termed SKI-606 179 is currently being evaluated in phase 1 studies, and others, such as NS-187 (INNO406), 180 AZD0530, 181 PD166326, 182 and PD180970, 183 may soon follow.…”