Differential ability of Tribbles family members to promote degradation of C/EBPalpha and induce acute myelogenous leukemia.

Trib1, Trib2, and Trib3 are mammalian homologs of Tribbles, an evolutionarily conserved Drosophila protein family that mediates protein degradation. Tribbles proteins function as adapters to recruit E3 ubiquitin ligases and enhance ubiquitylation of the target protein to promote its degradation. Increased Trib1 and Trib2 mRNA expression occurs in human myeloid leukemia and induces acute myeloid leukemia in mice, whereas Trib3 has not been associated with leukemia. Given the high degree of structural conservation among Tribbles family members, we directly compared the 3 mammalian Tribbles in hematopoietic cells by reconstituting mice with hematopoietic stem cells retrovirally expressing these proteins. All mice receiving Trib1 or Trib2 transduced hematopoietic stem cells developed acute myeloid leukemia, whereas Trib3 mice did not. Our previous data indicated that Trib2-mediated degradation of the transcription factor, CCAAT/enhancer-binding protein-alpha (C/EBPalpha), is important for leukemogenesis. Similar to Trib2, Trib1 induced C/EBPalpha degradation and inhibited its function. In contrast, Trib3 failed to inactivate or promote efficient degradation of C/EBPalpha. These data reveal that the 3 Tribbles homologs differ in their ability to promote degradation of C/EBPalpha, which account for their differential ability to induce leukemia.

Leukemia-associated NOTCH1 alleles are weak tumor initiators but accelerate K-ras-initiated leukemia.

Gain-of-function NOTCH1 mutations are found in 50%-70% of human T cell acute lymphoblastic leukemia/lymphoma (T-ALL) cases. Gain-of-function NOTCH1 alleles that initiate strong downstream signals induce leukemia in mice, but it is unknown whether the gain-of-function NOTCH1 mutations most commonly found in individuals with T-ALL generate downstream signals of sufficient strength to induce leukemia. We addressed this question by expressing human gain-of-function NOTCH1 alleles of varying strength in mouse hematopoietic precursors. Uncommon gain-of-function NOTCH1 alleles that initiated strong downstream signals drove ectopic T cell development and induced leukemia efficiently. In contrast, although gain-of-function alleles that initiated only weak downstream signals also induced ectopic T cell development, these more common alleles failed to efficiently initiate leukemia development. However, weak gain-of-function NOTCH1 alleles accelerated the onset of leukemia initiated by constitutively active K-ras and gave rise to tumors that were sensitive to Notch signaling pathway inhibition. These data show that induction of leukemia requires doses of Notch1 greater than those needed for T cell development and that most NOTCH1 mutations found in T-ALL cells do not generate signals of sufficient strength to initiate leukemia development. Furthermore, low, nonleukemogenic levels of Notch1 can complement other leukemogenic events, such as activation of K-ras. Even when Notch1 participates secondarily, the resulting tumors show "addiction" to Notch, providing a further rationale for evaluating Notch signaling pathway inhibitors in leukemia.

Loss of the mismatch repair protein MSH6 in human glioblastomas is associated with tumor progression during temozolomide treatment.

PURPOSE: Glioblastomas are treated by surgical resection followed by radiotherapy [X-ray therapy (XRT)] and the alkylating chemotherapeutic agent temozolomide. Recently, inactivating mutations in the mismatch repair gene MSH6 were identified in two glioblastomas recurrent post-temozolomide. Because mismatch repair pathway inactivation is a known mediator of alkylator resistance in vitro, these findings suggested that MSH6 inactivation was causally linked to these two recurrences. However, the extent of involvement of MSH6 in glioblastoma is unknown. We sought to determine the overall frequency and clinical relevance of MSH6 alterations in glioblastomas. EXPERIMENTAL DESIGN: The MSH6 gene was sequenced in 54 glioblastomas. MSH6 and O(6)-methylguanine methyltransferase (MGMT) immunohistochemistry was systematically scored in a panel of 46 clinically well-characterized glioblastomas, and the corresponding patient response to treatment evaluated. RESULTS: MSH6 mutation was not observed in any pretreatment glioblastoma (0 of 40), whereas 3 of 14 recurrent cases had somatic mutations (P = 0.015). MSH6 protein expression was detected in all pretreatment (17 of 17) cases examined but, notably, expression was lost in 7 of 17 (41%) recurrences from matched post-XRT + temozolomide cases (P = 0.016). Loss of MSH6 was not associated with O(6)-methylguanine methyltransferase status. Measurements of in vivo tumor growth using three-dimensional reconstructed magnetic resonance imaging showed that MSH6-negative glioblastomas had a markedly increased rate of growth while under temozolomide treatment (3.17 versus 0.04 cc/mo for MSH6-positive tumors; P = 0.020). CONCLUSIONS: Loss of MSH6 occurs in a subset of post-XRT + temozolomide glioblastoma recurrences and is associated with tumor progression during temozolomide treatment, mirroring the alkylator resistance conferred by MSH6 inactivation in vitro. MSH6 deficiency may therefore contribute to the emergence of recurrent glioblastomas during temozolomide treatment.