Kinase Domain Mutants of Bcr-Abl Exhibit Altered Transformation Potency, Kinase Activity, and Substrate Utilization, Irrespective of Sensitivity to Imatinib

Abstract

Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gainor loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.Bcr-Abl, a constitutively active tyrosine kinase, is the defining molecular feature of chronic myeloid leukemia (CML) (8). Biochemical studies and murine models have established that tyrosine kinase activity is essential to the transforming capacity of Bcr-Abl (7, 17). Consistent with this, inhibition of the BcrAbl kinase with imatinib, an Abl-specific kinase inhibitor, induces remissions in patients with CML (9, 10). Although responses in the early phases of CML are frequently durable, relapse is common in patients with advanced-phase CML. Mutations in the kinase domain (KD) of Bcr-Abl that impair imatinib binding have been identified as the major mechanism of acquired resistance (1,3,13,15,34,37). However, in some patients, drug-resistant mutants were detected prior to therapy (16,27,28,34,38). The proportion of mutant allele in these samples varied, from being detectable only with allele-specific PCR, which could detect as few as one cell in 100,000 (38), to representing 40% of the BCR-ABL message (34). These data suggest that some mutants may have a proliferative advantage over unmutated Bcr-Abl (herein referred to as native Bcr-Abl) even in the absence of imatinib, while others may be loss-offunction alleles that are selected only in the presence of imatinib. Mutations of the ATP binding loop (P-loop) of Abl are associated with significantly shorter survival than other mutations, regardless of their sensitivity to imatinib (3, 36), suggesting that certain Bcr-Abl mutations may directly contribute to disease progression by conferring a more aggressive phenotype. To test these hypotheses, we performed various biochemical and biological assays to compare five common KD mutants that comprise ca. 60% of Bcr-Abl m...

Prevalence and dynamics of bcr-abl kinase domain mutations during imatinib treatment differ in patients with newly diagnosed and recurrent bcr-abl positive acute lymphoblastic leukemia

“…The majority of patients with Ph þ ALL who relapse during the treatment with a TKI carry bcr-abl TKD mutations that display high IC 50 values in biochemical and cellular assays, 13,20,21 implying that they have a causal role in acquired imatinib resistance. 12 --19 Their role in primary resistance commonly observed in Ph þ ALL recurring after chemotherapy is uncertain, and it also remains to be determined when these mutations first develop.…”

“…Conceptually, the inferior imatinib response observed in these patients with advanced Ph þ ALL could be due to (i) a higher frequency of pre-existing mutations, (ii) a preponderance of mutations with greater transforming activity, 20 (iii) larger initial mutant clones or (iv) non-mutational resistance mechanisms. To evaluate the potential contributions of these mechanisms to primary resistance, we compared imatinib-naïve patients with newly diagnosed and recurrent Ph þ ALL in terms of the frequency and pattern of baseline mutations, the initial level of mutant clones, their outgrowth dynamics during the first 4 weeks of imatinib monotherapy and the relation between mutational status and time to progression (TTP).…”

Bortezomib decreases Rb phosphorylation and induces caspase-dependent apoptosis in Imatinib-sensitive and -resistant Bcr-Abl1-expressing cells

“…BaF/3 control cells, Bcr-Abl1-expressing BaF/3-p210 cells, and BaF/3 cells expressing IM-resistant Bcr-Abl1 mutants were a kind gift of Dr Druker (Griswold et al, 2006). The TCC-S cell line derived from a CML patient in blast crisis and that expresses mainly the p210 form of Bcr-Abl1 and, to a lower extent, also the p190 form of the oncoprotein (Van et al, 2005) was obtained from the American Type Culture Collection (Manassas, VA, USA).…”

Allosterische Kinaseinhibitoren

“…Dies ist insbesondere verwunderlich, wenn man bedenkt, wie weit diese Mutation von der Myristat-Bindungstasche entfernt ist und daher nicht sterisch mit der GNF-2-Bindung interferieren kann. Auf der anderen Seite wurde aber auch gezeigt dass einige Imatinib-Resistenzmutationen, insbesondere T315I, Gain-of-functionEigenschaften haben und sowohl die enzymatische Aktivität als auch onkogene Transformation durch BCR-ABL1 erhöhen können [2,6,21]. Daher wurde argumentiert, dass diese Mutationen eine aktivere Konformation der ABLKinase-Domäne stablisieren könnten, an die Imatinib weniger gut binden kann.…”

“…Des Weiteren konnte [2,6,21] gezeigt werden, dass die Kombination von GNF-5 mit dem Zweitgenerationsinhibitor Nilotinib das Überleben in einem Maus-Xenotransplantationsmodell mit BCR-ABL1-T315I-exprimierenden Zellen stark verlängert [24]. In diesem Modell zeigt die Einzeltherapie mit beiden Medikamenten keine Wirkung.…”