Eric P. Stoffregen

In vitro Activity of Bcr-Abl Inhibitors AMN107 and BMS-354825 against Clinically Relevant Imatinib-Resistant Abl Kinase Domain Mutants

Imatinib, a Bcr-Abl tyrosine kinase inhibitor, is a highly effective therapy for patients with chronic myelogenous leukemia (CML). Despite durable responses in most chronic phase patients, relapses have been observed and are much more prevalent in patients with advanced disease. The most common mechanism of acquired imatinib resistance has been traced to Bcr-Abl kinase domain mutations with decreased imatinib sensitivity. Thus, alternate Bcr-Abl kinase inhibitors that have activity against imatinib-resistant mutants would be useful for patients who relapse on imatinib therapy. Two such Bcr-Abl inhibitors are currently being evaluated in clinical trials: the improved potency, selective Abl inhibitor AMN107 and the highly potent dual Src/Abl inhibitor BMS-354825. In the current article, we compared imatinib, AMN107, and BMS-354825 in cellular and biochemical assays against a panel of 16 kinase domain mutants representing >90% of clinical isolates. We report that AMN107 and BMS-354825 are 20-fold and 325-fold more potent than imatinib against cells expressing wild-type Bcr-Abl and that similar improvements are maintained for all imatinib-resistant mutants tested, with the exception of T315I. Thus, both inhibitors hold promise for treating imatinib-refractory CML.

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 gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.

Combined Abl Inhibitor Therapy for Minimizing Drug Resistance in Chronic Myeloid Leukemia: Src/Abl Inhibitors Are Compatible with Imatinib

Purpose: Chronic myeloid leukemia (CML) is effectively treated with imatinib. However, reactivation of Bcr-Abl via kinase domain mutations that reduce sensitivity to imatinib can cause relapse. As combination therapy is frequently used to prevent emergence of resistance, the combination of imatinib with an inhibitor of imatinib-resistant Bcr-Abl mutants (e.g., Src/Abl inhibitors AP23848 and BMS-354825) was investigated. Experimental Design: To test this approach, cellular proliferation and Bcr-Abl tyrosine phosphorylation assays were done on Ba/F3 cells expressing wild-type (WT) Bcr-Abl and four common imatinib-resistant mutants (Y253F, E255K, T315I, and M351T). Colony-forming assays with primary CML cells were also done. Results: Both Src/Abl inhibitors retained full inhibitory capacity when coadministered with imatinib at concentrations above typical clinical levels. For cells expressing WT Bcr-Abl or the marginally imatinib-resistant mutant M351T, inclusion of imatinib at therapeutic levels enhanced the effects of the Src/Abl inhibitors. By comparison, for the highly imatinib-resistant mutants Y253F and E255K, inclusion of imatinib at clinical levels resulted in only a slight enhancement beyond the effects of the Src/Abl inhibitors. None of the inhibitors affected Bcr-Abl T315I cells. Colony-forming assays with primary CML cells yielded analogous results. Conclusions: Our results indicate that Src/Abl inhibitors are compatible with imatinib and suggest that combined Abl inhibitor therapy is a feasible treatment strategy for patients with CML.

An autosomal locus that controls chromosome-wide replication timing and mono-allelic expression

Mammalian DNA replication initiates at multiple sites along chromosomes at different times, following a temporal replication program. Homologous alleles typically replicate synchronously; however, mono-allelically expressed genes such as imprinted genes, allelically excluded genes and genes on the female X chromosome replicate asynchronously. We have used a chromosome engineering strategy to identify a human autosomal locus that controls this replication timing program in cis. We show that Cre/loxP-mediated rearrangements at a discrete locus at 6q16.1 result in delayed replication of the entire chromosome. This locus displays asynchronous replication timing that is coordinated with other mono-allelically expressed genes on chromosome 6. Characterization of this locus revealed mono-allelic expression of a large intergenic non-coding RNA, which we have named asynchronous replication and autosomal RNA on chromosome 6, ASAR6. Finally, disruption of this locus results in the activation of the previously silent alleles of linked mono-allelically expressed genes. We previously found that chromosome rearrangements involving eight different autosomes display delayed replication timing, and that cells containing chromosomes with delayed replication timing have a 30-80-fold increase in the rate at which new gross chromosomal rearrangements occurred. Taken together, these observations indicate that human autosomes contain discrete cis-acting loci that control chromosome-wide replication timing, mono-allelic expression and the stability of entire chromosomes.

Asynchronous Replication, Mono-Allelic Expression, and Long Range Cis-Effects of ASAR6

Mammalian chromosomes initiate DNA replication at multiple sites along their length during each S phase following a temporal replication program. The majority of genes on homologous chromosomes replicate synchronously. However, mono-allelically expressed genes such as imprinted genes, allelically excluded genes, and genes on female X chromosomes replicate asynchronously. We have identified a cis-acting locus on human chromosome 6 that controls this replication-timing program. This locus encodes a large intergenic non-coding RNA gene named Asynchronous replication and Autosomal RNA on chromosome 6, or ASAR6. Disruption of ASAR6 results in delayed replication, delayed mitotic chromosome condensation, and activation of the previously silent alleles of mono-allelic genes on chromosome 6. The ASAR6 gene resides within an ∼1.2 megabase domain of asynchronously replicating DNA that is coordinated with other random asynchronously replicating loci along chromosome 6. In contrast to other nearby mono-allelic genes, ASAR6 RNA is expressed from the later-replicating allele. ASAR6 RNA is synthesized by RNA Polymerase II, is not polyadenlyated, is restricted to the nucleus, and is subject to random mono-allelic expression. Disruption of ASAR6 leads to the formation of bridged chromosomes, micronuclei, and structural instability of chromosome 6. Finally, ectopic integration of cloned genomic DNA containing ASAR6 causes delayed replication of entire mouse chromosomes.

A single nucleotide polymorphism in the coding region of ABL and its effects on sensitivity to imatinib

We have identified a gene polymorphism (K247R) within or close to the P-loop of BCR-ABL, which leads to the substitution of arginine for lysine. We investigated the allelic frequency of K247R by screening 157 CML patients and 213 healthy blood donors with conventional sequencing, restriction enzyme digest and single strand conformational polymorphism analysis, and found the arginine allele to be rare. Three out of five CML patients with the arginine allele of K247R failed to achieve a major cytogenetic response to imatinib, suggesting that the arginine allele may have reduced sensitivity. However, despite K247Rs position in or near to the P-loop, biochemical and cellular assays of imatinib and dasatinib sensitivity showed no alteration compared to wild type. Clinicians should be aware that possession of the arginine allele of K247R does not reflect a mutation that necessitates a change in the therapeutic strategy, unless there are other signs of inadequate response to drug.