Deanne Tibbitts

FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to γ-secretase inhibitors

γ-secretase inhibitors (GSIs) can block NOTCH receptor signaling in vitro and therefore offer an attractive targeted therapy for tumors dependent on deregulated NOTCH activity. To clarify the basis for GSI resistance in T cell acute lymphoblastic leukemia (T-ALL), we studied T-ALL cell lines with constitutive expression of the NOTCH intracellular domain (NICD), but that lacked C-terminal truncating mutations in NOTCH1. Each of the seven cell lines examined and 7 of 81 (8.6%) primary T-ALL samples harbored either a mutation or homozygous deletion of the gene FBW7, a ubiquitin ligase implicated in NICD turnover. Indeed, we show that FBW7 mutants cannot bind to the NICD and define the phosphodegron region of the NICD required for FBW7 binding. Although the mutant forms of FBW7 were still able to bind to MYC, they do not target it for degradation, suggesting that stabilization of both NICD and its principle downstream target, MYC, may contribute to transformation in leukemias with FBW7 mutations. In addition, we show that all seven leukemic cell lines with FBW7 mutations were resistant to the MRK-003 GSI. Most of these resistant lines also failed to down-regulate the mRNA levels of the NOTCH targets MYC and DELTEX1 after treatment with MRK-003, implying that residual NOTCH signaling in T-ALLs with FBW7 mutations contributes to GSI resistance.

The Axin1 scaffold protein promotes formation of a degradation complex for c‐Myc

Expression of the c‐Myc proto‐oncoprotein is tightly regulated in normal cells. Phosphorylation at two conserved residues, threonine58 (T58) and serine62 (S62), regulates c‐Myc protein stability. In cancer cells, c‐Myc can become aberrantly stabilized associated with altered T58 and S62 phosphorylation. A complex signalling cascade involving GSK3β kinase, the Pin1 prolyl isomerase, and the PP2A‐B56α phosphatase controls phosphorylation at these sites. We report here a novel role for the tumour suppressor scaffold protein Axin1 in facilitating the formation of a degradation complex for c‐Myc containing GSK3β, Pin1, and PP2A‐B56α. Although knockdown of Axin1 decreases the association of c‐Myc with these proteins, reduces T58 and enhances S62 phosphorylation, and increases c‐Myc stability, acute expression of Axin1 reduces c‐Myc levels and suppresses c‐Myc transcriptional activity. Moreover, the regulation of c‐Myc by Axin1 is impaired in several tested cancer cell lines with known stabilization of c‐Myc or loss of Axin1. This study provides critical insight into the regulation of c‐Myc expression, how this can be disrupted in three cancer types, and adds to our knowledge of the tumour suppressor activity of Axin1.

Aberrant stabilization of c-Myc protein in some lymphoblastic leukemias

Overexpression of the c-Myc oncoprotein is observed in a large number of hematopoietic malignancies, and transgenic animal models have revealed a potent role for c-Myc in the generation of leukemias and lymphomas. However, the reason for high c-Myc protein levels in most cases is unknown. We examined whether aberrant protein stabilization could be a mechanism of c-Myc overexpression in leukemia cell lines and in primary bone marrow samples from pediatric acute lymphoblastic leukemia (ALL) patients. We found that c-Myc protein half-life was prolonged in the majority of leukemia cell lines and bone marrow samples tested. There were no mutations in the c-myc gene in any of the leukemia cell lines that could account for increased c-Myc stability. However, abnormal phosphorylation at two conserved sites, Threonine 58 and Serine 62, was observed in leukemia cell lines with stabilized c-Myc. Moreover, stabilized c-Myc from the ALL cell lines showed decreased affinity for glycogen synthase kinase3β, the kinase that phosphorylates c-Myc at Threonine 58 and facilitates its degradation. These findings reveal that deregulation of the c-Myc degradation pathway controlled by Serine 62 and Threonine 58 phosphorylation is a novel mechanism for increased expression of a potent oncoprotein known to be involved in hematopoietic malignancies.

Combined targeting of SET and tyrosine kinases provides an effective therapeutic approach in human T-cell acute lymphoblastic leukemia

Recent evidence suggests that inhibition of protein phosphatase 2A (PP2A) tumor suppressor activity via the SET oncoprotein contributes to the pathogenesis of various cancers. Here we demonstrate that both SET and c-MYC expression are frequently elevated in T-ALL cell lines and primary samples compared to healthy T cells. Treatment of T-ALL cells with the SET antagonist OP449 restored the activity of PP2A and reduced SET interaction with the PP2A catalytic subunit, resulting in a decrease in cell viability and c-MYC expression in a dose-dependent manner. Since a tight balance between phosphatases and kinases is required for the growth of both normal and malignant cells, we sought to identify a kinase inhibitor that would synergize with SET antagonism. We tested various T-ALL cell lines against a small-molecule inhibitor screen of 66 compounds targeting two-thirds of the tyrosine kinome and found that combined treatment of T-ALL cells with dovitinib, an orally active multi-targeted small-molecule receptor tyrosine kinase inhibitor, and OP449 synergistically reduced the viability of all tested T-ALL cell lines. Mechanistically, combined treatment with OP449 and dovitinib decreased total and phospho c-MYC levels and reduced ERK1/2, AKT, and p70S6 kinase activity in both NOTCH-dependent and independent T-ALL cell lines. Overall, these results suggest that combined targeting of tyrosine kinases and activation of serine/threonine phosphatases may offer novel therapeutic strategies for the treatment of T-ALL.

Studying c-Myc serine 62 phosphorylation in leukemia cells: Concern over antibody cross-reactivity

To the editor: A recent article in Blood demonstrated that the Pim kinase inhibitor SGI-1776 has efficacy in acute myeloid leukemia.[1][1] This inhibitor reduced phosphorylation of multiple targets of the Pim kinases, including c-Myc at serine 62 (S62). Because no therapies directly target c-Myc,

Abstract A28: Aberrant c‐Myc protein stabilization and Axin1 deregulation in acute leukemia

The c‐Myc oncoprotein is an important regulator of cellular growth, proliferation, differentiation, and apoptosis, and c‐Myc expression is commonly increased in cancer. Historically, increased c‐Myc expression in hematopoietic cancers has been explained by gene amplification or chromosomal translocations that deregulate its transcription. Recent work from our lab suggests that deregulated phosphorylation of c‐Myc associated with disrupted c‐Myc degradation constitutes an additional mechanism by which c‐Myc can drive cancer. We have previously shown that c‐Myc degradation is regulated by hierarchical phosphorylation at 2 conserved sites, serine 62 (S62) and threonine 58 (T58), and is coordinated by the scaffold protein and tumor suppressor Axin1. Building on our previous work showing that in a variety of human leukemia cell lines c‐Myc is aberrantly stabilized with altered phosphorylation at T58 and S62, we are examining the prevalence and potential mechanism of increased c‐Myc stability in primary acute myelogenous leukemia (AML), T cell acute lymphoblastic leukemia (ALL), and B cell ALL patient samples. Based on our recent work showing that c‐Myc protein stability is negatively regulated by Axin1, we are examining mechanisms of Axin1 deregulation in AML and ALL patient samples. Finally, by manipulating Axin1 in both leukemic and non‐transformed hematopoietic cell lines, we will investigate Axin19s role in directly controlling c‐Myc stability and phosphorylation in hematopoietic lineages and whether Axin1 may be a potential therapeutic target in acute leukemias. Citation Information: Cancer Res 2009;69(23 Suppl):A28.