Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction

Abstract

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...

Apoptosis-based dual molecular targeting by INNO-406, a second-generation Bcr-Abl inhibitor, and ABT-737, an inhibitor of antiapoptotic Bcl-2 proteins, against Bcr-Abl-positive leukemia

“…29,30 Furthermore, Bcr-Abl inhibitors have been shown to be more cytostatic than cytotoxic against Bcr-Abl þ leukemic cells. 9,10 These suggest that the complete eradication of Bcr-Abl þ leukemic cells may require Bcr-Abl inhibitors in combination with therapies that modify the molecular pathways that control cell survival. Our study supports this premise, as we were able to markedly increase the killing of transcriptional activator of bim.…”

“…7,8 However, it appears that a few leukemic cells often survive exposure to imatinib or second-generation Bcr-Abl inhibitors. 9,10 Indeed, mathematical modeling studies suggest that complete eradication of Bcr-Abl þ leukemic cells may require simultaneous targeting of other vital molecules. 11,12 Here, we have sought to identify additional therapeutic targets by characterizing the molecular mechanism by which the blockade of Bcr-Abl signaling kills Ph þ leukemia cells.…”

ReSETting PP2A tumour suppressor activity in blast crisis and imatinib-resistant chronic myelogenous leukaemia

“…The development of the BCR/ABL tyrosine kinase inhibitor imatinib mesylate (Gleevec; formerly STI571) as the treatment of choice for chronic phase CML and its remarkable therapeutic effects suggest that blast crisis transition will be postponed for several years in the majority of CML patients (Deininger et al, 2005a;Roy et al, 2006). However, the persistence of BCR/ABL transcripts in a cohort of patients with complete cytogenetic response (Hughes et al, 2003) and the resistance of the primitive CML stem cell to imatinib treatment (Copland et al, 2006) raises the possibility that treatment with imatinib alone might delay but not prevent disease progression. Furthermore, most of the CML patients in the accelerated and blastic phases of the disease are either refractory or develop resistance to imatinib monotherapy (Deininger et al, 2005a).…”

“…Although new phase 1 clinical trials with the dual Src/Abl inhibitor dasatinib (BMS-354825) and the selective Abl inhibitor AMN107 show encouraging results (O'Hare et al, 2005), as they suppress the activity of most BCR/ABL mutants (except T315I) (O'Hare et al, 2005), in vitro evidence suggests that resistance to these new compounds may develop through mechanisms involving the selection and expansion of BCR/ABL þ cell clones carrying the T315I BCR/ABL mutant (Deininger et al, 2005b). Additionally, dasatinib, like imatinib, is not effective in the treatment of CML-BC patients (Talpaz et al, 2006), and in killing the most primitive quiescent CML cells (Copland et al, 2006) and, therefore, it may also be ineffective in preventing disease progression.…”

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

“…For myeloproliferative neoplasms (MPN) and leukaemias, this stem cell population has been associated with deregulated protein tyrosine kinase (PTK) activity (for review see Mitelman 10 ), including FMS-like tyrosine kinase 3 (FLT3) in acute myeloid leukaemia (AML) 11 , Janus kinase 2 (JAK2) in erythroid neoplasia 12 and BCR-ABL in chronic myeloid leukaemia (CML) 13,14 . Tyrosine kinase inhibitor (TKI) therapy using imatinib mesylate (IM) has been shown to inhibit PTK activity in CML [15][16][17][18] . The use of TKIs has improved clinical outcomes markedly for the majority of patients with chronic phase (CP) CML who achieve sustained cytogenetic and molecular response 19,20 .…”