Dasatinib (BMS-354825), a novel dual SRC/BCR-ABL kinase inhibitor, exhibits greater potency than imatinib mesylate (IM) and inhibits the majority of kinase mutations in IM-resistant chronic myeloid leukemia (CML). We have previously demonstrated that IM reversibly blocks proliferation but does not induce apoptosis of primitive CML cells. Here, we have attempted to overcome this resistance with dasatinib. Primitive IM-resistant CML cells showed only single-copy BCR-ABL but expressed significantly higher BCR-ABL transcript levels and BCR-ABL protein compared with more mature CML cells (P ‫؍‬ .031). In addition, CrKL phosphorylation was higher in the primitive CD34 ؉ CD38 ؊ than in the total CD34 ؉ population (P ‫؍‬ .002). In total CD34 ؉ CML cells, IM inhibited phosphorylation of CrKL at 16 but not 72 hours, consistent with enrichment of an IM-resistant primitive population. CD34 ؉ CD38 ؊ CML cells proved resistant to IM-induced inhibition of CrKL phosphorylation and apoptosis, whereas dasatinib led to significant inhibition of CrKL phosphorylation. Kinase domain mutations were not detectable in either IM or dasatinib-resistant primitive CML cells. These data confirm that dasatinib is more effective than IM within the CML stem cell compartment; however, the most primitive quiescent CML cells appear to be inherently resistant to both drugs. ( IntroductionChronic myeloid leukemia (CML) is a clonal hemopoietic disorder that is sustained by a population of primitive and transplantable stem cells. 1 These stem cells are Philadelphia chromosome positive (Ph ϩ ) and express the oncogenic tyrosine kinase BCR-ABL. 2,3 BCR-ABL is central to the pathogenesis of CML, and mutation of critical elements leads to a reduction in transformation potential. 4,5 In the malignant clone, BCR-ABL is constitutively active, resulting in autophosphorylation of the kinase domain and of downstream substrates including CrKL. 6,7 The specificity of CrKL phosphorylation to BCR-ABL signaling, partnered with stability of the phosphoprotein complex, has led to its acceptance as an excellent method to assess BCR-ABL status. [8][9][10] Imatinib mesylate (IM) has been introduced as first-line targeted therapy for CML. IM is a tyrosine kinase inhibitor that is relatively specific for BCR-ABL. 11 In vitro, in Ph ϩ cell lines and in bulk cultures of primary CML cells, IM reverses the autophosphorylation of BCR-ABL and inhibits phosphorylation of downstream targets including CrKL. 11,12 Within 48 to 72 hours of IM exposure, CML cells undergo apoptosis. More recently, Chu et al 13 have confirmed that CrKL phosphorylation is also inhibited by IM in CD34-enriched populations of primary CML cells. However, our own work and that of others confirms that primitive CML cells do not readily undergo apoptosis, even after prolonged in vitro exposure to the drug. [14][15][16][17] The link between inactivation of BCR-ABL kinase activity and induction of apoptosis in the most primitive CML cells therefore remains unclear.In vivo, even in chronic phase, clinical respon...

Background: Pancreatic cancer is one of the most deadly malignancies with insufficient therapeutic options and poor outcome. Cancer stem cells (CSCs) are thought to be responsible for progression and therapy resistance. We investigated the potential of pancreatic cell lines for CSC research by analyzing to what extent they contain CSC populations and how representative these are compared to clinical tissue. Methods: Six pancreatic cancer cell lines were analyzed by flow cytometry for CD326, CD133, CD44, CD24, CXCR4 and ABCG2. Subsequently, 70 primary pancreatic tissues were evaluated for CD326, CD133 and CD44 by immunohistochemistry. Results: All the cell lines but one showed a stable expression pattern throughout biological replicates. Marker expression in clinical tissue of CD44 distinguished normal patients from pancreatic carcinoma patients with a sensitivity of 50% at 80% specificity and metastasized from nonmetastasized carcinomas with 69% sensitivity at 100% specificity. Conclusions: Our results indicate a link between elevated CD44 expression, malignancy and metastasis of pancreatic tissue. Furthermore, individual pancreatic cell lines show a substantial amount of cells with CSC properties which is comparable with interpatient variability detected in primary tissue. These pancreatic cancer cell lines could thus serve for urgently needed pharmacological CSC in vitro research.

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.