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...

In chronic-phase chronic myeloid leukemia (CML) patients, the lack of a major cytogenetic response (< 36% Ph ؉ metaphases) to imatinib within 12 months indicates failure and mandates a change of therapy. To identify biomarkers predictive of imatinib failure, we performed gene expression array profiling of CD34 ؉ cells from 2 independent cohorts of imatinib-naive chronic-phase CML patients. The learning set consisted of retrospectively selected patients with a complete cytogenetic response or more than 65% Ph ؉ metaphases within 12 months of imatinib therapy. Based on analysis of variance P less than .1 and fold difference 1.5 or more, we identified 885 probe sets with differential expression between responders and nonresponders, from which we extracted a 75-probe set minimal signature (classifier) that separated the 2 groups. On application to a prospectively accrued validation set, the classifier correctly predicted 88% of responders and 83% of nonresponders. Bioinformatics analysis and comparison with published studies revealed overlap of classifier genes with CML progression signatures and implicated ␤-catenin in their regulation, suggesting that chronic-phase CML patients destined to fail imatinib have more advanced disease than evident by morphologic criteria. Our classifier may allow directing more aggressive therapy upfront to the patients most likely to benefit while sparing good-risk patients from unnecessary toxicity. (Blood. 2010;115:315-325)

Aberrant MYC gene expression by the Wnt/β-catenin pathway is implicated in colorectal carcinogenesis. Wnt/β-catenin signaling stimulates association of the β-catenin coactivator complex with two Wnt responsive enhancers (WREs) located in close proximity to MYC gene boundaries. Each enhancer directly binds members of the TCF/Lef family of transcription factors that, in turn, recruit β-catenin. In a previous report, we showed that the downstream MYC enhancer (MYC 3′ WRE) cooperated with the upstream enhancer (MYC 5′ WRE) to activate expression of a heterologous reporter gene in response to Wnt/β-catenin and mitogen signaling. Here we use chromatin conformation capture (3C) to show that the MYC 5′ and 3′ WREs are juxtaposed at the genomic MYC locus during active transcription. This MYC 5′3′ chromatin loop is present in HCT116 human colorectal cancer cells that contain high levels of nuclear β-catenin and is absent in HEK293 cells that contain trace amounts of nuclear β-catenin. Depletion of functional β-catenin/TCF complexes blocks formation of the MYC 5′3 chromatin loop. Furthermore, we find that the chromatin loop is absent in quiescent cells, but is rapidly and transiently induced by serum mitogens in a β-catenin-dependent manner. Thus, we propose that a distinct chromatin architecture coordinated by β-catenin/TCF-bound WREs accompanies transcriptional activation of MYC gene expression.colon cancer | chromatin conformation capture T he Wnt/β-catenin signaling pathway plays a critical role in regulating cell proliferation, cell migration, and stem cell self-renewal in the gastrointestinal tract (1). β-Catenin is the key mediator of cellular responses to Wnt signaling, and its cytoplasmic/nuclear partitioning is tightly controlled. When Wnt is unavailable, cytoplasmic β-catenin associates with a multiprotein destruction complex that targets its degradation via the proteasome. Thus, in the absence of Wnt ligand, there is little, if any, nuclear β-catenin and Wnt/β-catenin target genes are repressed by CtBP and Groucho/TLE corepressor complexes (2). These corepressors are tethered to target genes through interactions with members of the T-cell factor/lymphoid enhancer binding factor (TCF/Lef) family of transcription factors. In the presence of Wnt, cytoplasmic β-catenin is stabilized and subsequently translocated to the nucleus. Nuclear β-catenin binds to TCF/Lef, displaces the corepressor complexes, and recruits coactivator complexes including CBP/p300 acetyltransferases and MLL/Set methyltransferases to activate gene expression (2).Mutations in components of the Wnt/β-catenin pathway are among the earliest detected lesions during colorectal carcinogenesis (3). In most cases of colorectal cancer, missense mutations target the adenomatous polyposis coli (APC) gene and lead to synthesis of a truncated APC protein. Truncated APC has diminished capacity to coordinate degradation of cytoplasmic β-catenin and, as a result, β-catenin inappropriately accumulates in the nuclei of colonic epithelial and stem cells. Therefore...

The transcriptional co-activator p300 has been reported to regulate the tumor suppressor p53 and its ortholog p73. Here we describe a study showing that this coactivator also regulates the transcriptional function of p63. p300 bound to the N-terminal domain of p63␥, and p63␥ bound to the N terminus of p300 in vitro and in cells. p300, but not its acetylase-defective mutant AT2, stimulated p63␥-dependent transcription and induction of p21 in cells, consequently leading to G 1 arrest. Inversely, the ⌬N-p63␥ isoform as well as p300AT2 inhibited the induction of p21 by p63␥. These results suggest that p300 regulates p63-dependent transcription of p21.

Post-translational modification of the p53 family members is key to their regulation. Here we report the phosphorylation of TAp63␥, but not ⌬Np63␥, by IB kinase ␤ (IKK␤). Activation of IKK␤ by ␥ radiation or tumor necrosis factor-␣ led to increased TAp63␥ protein levels in cells. IKK␤, but not its kinase-defective mutant IKK␤-K44A, led to this observed stabilization of TAp63␥. This stabilization of TAp63␥ in response to ␥ radiation was significantly decreased in the absence of IKK␤. Phosphorylation of TAp63␥ blocks ubiquitylation and possible degradation of this protein. We postulate that phosphorylation of TAp63␥ by IKK␤ stabilizes the TAp63␥ protein by blocking ubiquitylation-dependent degradation of this protein.