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...

Chronic myeloid leukemia (CML) stem and progenitor cells overexpress BcrAbl and are insensitive to imatinib mesylate (IM). We therefore investigated whether these cells were efficiently targeted by nilotinib. In K562, the inhibitory concentration (IC50) of nilotinib was 30 nM versus 600 nM for IM, consistent with its reported 20-fold-higher potency. However, in primary CD34(+) CML cells, nilotinib and IM were equipotent for inhibition of BcrAbl activity, producing equivalent but incomplete reduction in CrkL phosphorylation at 5 microM. CML CD34(+) cells were still able to expand over 72 hours with 5 microM of either drug, although there was a concentration-dependent restriction of amplification. As for IM, the most primitive cells (CFSE(max)) persisted and accumulated over 72 hours with nilotinib and remained caspase-3 negative. Furthermore, nilotinib with IM led to further accumulation of this population, suggesting at least additive antiproliferative effects. These results confirmed that, like IM, the predominant effect of nilotinib is antiproliferative rather than proapoptotic.

Spinal cord injury (SCI) is a devastating condition with limited capacity for repair. Cell transplantation is a potential strategy to promote SCI repair with cells from the olfactory system being promising candidates. Although transplants of human olfactory mucosa (OM) are already ongoing in clinical trials, the repair potential of this tissue remains unclear. Previously, we identified mesenchymal-like stem cells that reside in the lamina propria (LP-MSCs) of rat and human OM. Little is known about these cells or their interactions with glia such as olfactory ensheathing cells (OECs), which would be co-transplanted with MSCs from the OM, or endogenous CNS glia such as oligodendrocytes. We have characterized, purified, and assessed the repair potential of human LP-MSCs by investigating their effect on glial cell biology with specific emphasis on CNS myelination in vitro. Purified LP-MSCs expressed typical bone marrow MSC (BM-MSC) markers, formed spheres, were clonogenic and differentiated into bone and fat. LP-MSC conditioned medium (CM) promoted oligodendrocyte precursor cell (OPC) and OEC proliferation and induced a highly branched morphology. LP-MSC-CM treatment caused OEC process extension. Both LP and BM-MSCs promoted OPC proliferation and differentiation, but only myelinating cultures treated with CM from LP and not BM-MSCs had a significant increase in myelination. Comparison with fibroblasts and contaminating OM fibroblast like-cells showed the promyelination effect was LP-MSC specific. Thus LP-MSCs harvested from human OM biopsies may be an important candidate for cell transplantation by contributing to the repair of SCI.