Runx transcription factors repress human and murine c-Myc expression in a DNA-binding and C-terminally dependent manner.

The transcription factors Runx1 and c-Myc have individually been shown to regulate important gene targets as well as to collaborate in oncogenesis. However, it is unknown whether there is a regulatory relationship between the two genes. In this study, we investigated the transcriptional regulation of endogenous c-Myc by Runx1 in the human T cell line Jurkat and murine primary hematopoietic cells. Endogenous Runx1 binds to multiple sites in the c-Myc locus upstream of the c-Myc transcriptional start site. Cells transduced with a C-terminally truncated Runx1 (Runx1.d190), which lacks important cofactor interaction sites and can block C-terminal-dependent functions of all Runx transcription factors, showed increased transcription of c-Myc. In order to monitor c-Myc expression in response to early and transiently-acting Runx1.d190, we generated a cell membrane-permeable TAT-Runx1.d190 fusion protein. Murine splenocytes treated with TAT-Runx1.d190 showed an increase in the transcription of c-Myc within 2 hours, peaking at 4 hours post-treatment and declining thereafter. This effect is dependent on the ability of Runx1.d190 to bind to DNA. The increase in c-Myc transcripts is correlated with increased c-Myc protein levels. Collectively, these data show that Runx1 directly regulates c-Myc transcription in a C-terminal- and DNA-binding-dependent manner.

Preclinical analysis of the γ-secretase inhibitor PF-03084014 in combination with glucocorticoids in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemias (T-ALL) and lymphomas are aggressive hematologic cancers frequently associated with activating mutations in NOTCH1. Early studies identified NOTCH1 as an attractive therapeutic target for the treatment of T-ALL through the use of γ-secretase inhibitors (GSI). Here, we characterized the interaction between PF-03084014, a clinically relevant GSI, and dexamethasone in preclinical models of glucocorticoid-resistant T-ALL. Combination treatment of the GSI PF-03084014 with glucocorticoids induced a synergistic antileukemic effect in human T-ALL cell lines and primary human T-ALL patient samples. Mechanistically PF-03084014 plus glucocorticoid treatment induced increased transcriptional upregulation of the glucocorticoid receptor and glucocorticoid target genes. Treatment with PF-03084014 and glucocorticoids in combination was highly efficacious in vivo, with enhanced reduction of tumor burden in a xenograft model of T-ALL. Finally, glucocorticoid treatment effectively reversed PF-03084014-induced gastrointestinal toxicity via inhibition of goblet cell metaplasia. These results warrant the analysis of PF-03084014 and glucocorticoids in combination for the treatment of glucocorticoid-resistant T-ALL.

Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we report the presence of loss-of-function mutations and deletions of the EZH2 and SUZ12 genes, which encode crucial components of the Polycomb repressive complex 2 (PRC2), in 25% of T-ALLs. To further study the role of PRC2 in T-ALL, we used NOTCH1-dependent mouse models of the disease, as well as human T-ALL samples, and combined locus-specific and global analysis of NOTCH1-driven epigenetic changes. These studies demonstrated that activation of NOTCH1 specifically induces loss of the repressive mark Lys27 trimethylation of histone 3 (H3K27me3) by antagonizing the activity of PRC2. These studies suggest a tumor suppressor role for PRC2 in human leukemia and suggest a hitherto unrecognized dynamic interplay between oncogenic NOTCH1 and PRC2 function for the regulation of gene expression and cell transformation.

Notch1 and TGFbeta1 cooperatively regulate Foxp3 expression and the maintenance of peripheral regulatory T cells.

Notch and its ligands have been implicated in the regulation and differentiation of various CD4(+) T-helper cells. Regulatory T cells (T(regs)), which express the transcription factor Foxp3, suppress aberrant immune responses that are typically associated with autoimmunity or excessive inflammation. Previous studies have shown that transforming growth factor beta (TGFbeta1) induces Foxp3 expression and a regulatory phenotype in peripheral T cells. Here, we show that pharmacologic inhibition of Notch signaling using gamma-secretase inhibitor (GSI) treatment blocks (1) TGFbeta1-induced Foxp3 expression, (2) the up-regulation of Foxp3-target genes, and (3) the ability to suppress naive T-cell proliferation. In addition, the binding of Notch1, CSL, and Smad to conserved binding sites in the foxp3 promoter can be inhibited by treatment with GSI. Finally, in vivo administration of GSI results in reduced Foxp3 expression and development of symptoms consistent with autoimmune hepatitis, a disease previously found to result from dysregulation of TGFbeta signaling and regulatory T cells. Together, these findings indicate that the Notch and TGFbeta signaling pathways cooperatively regulate Foxp3 expression and regulatory T-cell maintenance both in vitro and in vivo.