The interferon consensus sequence-binding protein (ICSBP/ IRF8) is an interferon regulatory factor that is expressed in myeloid and B-cells. ICSBP-deficient mice develop a myeloproliferative disorder characterized by cytokine hypersensitivity and apoptosis resistance. To identify ICSBP target genes involved in these effects, we screened a CpG island microarray with chromatin that co-immunoprecipitated with ICSBP from myeloid cells. Using this technique, we identified PTPN13 as an ICSBP target gene. PTPN13 encodes Fas-associated phosphatase 1 (Fap-1), a ubiquitously expressed protein-tyrosine phosphatase. This was of interest because interaction of Fap-1 with Fas results in Fas dephosphorylation and inhibition of Fas-induced apoptosis. In this study, we found that ICSBP influenced Fas-induced apoptosis in a Fap-1-dependent manner. We also found that ICSBP interacted with a cis element in the proximal PTPN13 promoter and repressed transcription. This interaction increased during myeloid differentiation and was regulated by phosphorylation of conserved tyrosine residues in the interferon regulatory factor domain of ICSBP. ICSBP deficiency was present in human myeloid malignancies, including chronic myeloid leukemia. Therefore, these studies identified a mechanism for increased survival of mature myeloid cells in the ICSBP-deficient murine model and in human myeloid malignancies with decreased ICSBP expression.

The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor, also referred to as IRF8. ICSBP acts as a suppressor of myeloid leukemia, although few target genes explaining this effect have been identified. In the current studies, we identified the gene encoding growth arrest specific 2 (GAS2) as an ICSBP target gene relevant to leukemia suppression. We find that ICSBP, Tel, and histone deacetylase 3 (HDAC3) bind to a cis element in the GAS2 promoter and repress transcription in myeloid progenitor cells. Gas2 inhibits calpain protease activity, and ␤-catenin is a calpain substrate in these cells. Consistent with this, ICSBP decreases ␤-catenin protein and activity in a Gas2-and calpain-dependent manner. Conversely, decreased ICSBP expression increases ␤-catenin protein and activity by the same mechanism. This is of interest, because decreased ICSBP expression and increased ␤-catenin activity are associated with poor prognosis and blast crisis in chronic myeloid leukemia (CML). We find that the expression of Bcr/abl (the CML oncoprotein) increases Gas2 expression in an ICSBP-dependent manner. This results in decreased calpain activity and a consequent increase in ␤-catenin activity in Bcr/abl-positive (Bcr/abl ؉ ) cells. Therefore, these studies have identified a Gas2/calpain-dependent mechanism by which ICSBP influences ␤-catenin activity in myeloid leukemia.

Transforming growth factor ␤ type II receptor (T␤RII) is a tumor suppressor gene that can be transcriptionally silenced by histone deacetylases (HDACs) in cancer cells. In this report, we demonstrated the mechanism by which trichostatin A (TSA), an inhibitor of HDAC, induces the expression of T␤RII in human pancreatic cancer cell lines by modulating the transcriptional components that bind a specific DNA region of the T␤RII promoter. This region of the T␤RII promoter possesses Sp1 and NF-Y binding sites in close proximity (located at ؊102 and ؊83, respectively). Treatment of cells with TSA activates the T␤RII promoter in a time-dependent manner through the recruitment of p300 and PCAF into a Sp1⅐NF-Y⅐HDAC complex that binds this DNA element. The recruitment of p300 and PCAF into the complex is associated with a concomitant acetylation of Sp1 and an overall decrease in the amount of HDAC associated with the complex. Transient overexpression of p300 or PCAF potentiated TSA-induced T␤RII promoter activity. The effect of PCAF was dependent on its histone acetyltransferase activity, whereas that of p300 was independent. Stable transfection of PCAF caused an increase in T␤RII mRNA expression, the association of PCAF with T␤RII promoter, and the acetylation of Sp1. Taken together, these results showed that TSA treatment of pancreatic cancer cells leads to transcriptional activation of the T␤RII promoter through modulation of the components of a Sp1⅐NF-Y⅐p300⅐PCAF⅐HDAC-1 multiprotein complex. Moreover, the interaction of NF-Y with the Sp1-associated complex may further explain why this specific Sp1 site mediates transcriptional responsiveness to TSA. TGF-␤1 plays a significant role in the growth inhibition of most normal epithelial and some cancer cells (1). TGF-␤ mediates its biological effects through cell surface receptors known as TGF-␤ type I receptor (T␤RI) and TGF-␤ type II receptor (T␤RII). Its intracellular signaling is initiated upon the selective binding of the active cytokine to the T␤RII homodimer. T␤RII is a ubiquitously expressed and constitutively active serine/threonine kinase. Ligand binding to T␤RII induces the assembly of a heterotetrameric complex consisting of T␤RI and T␤RII. Once the receptor complex is formed, T␤RII phosphorylates and thereby activates the T␤RI serine/threonine kinase. Activation of T␤RI propagates downstream signaling via Smad family proteins. T␤RI directly interacts with and phosphorylates Smad2 and Smad3. These Smads bind Smad4 and then result in the translocation of this complex to the nucleus and modulate TGF-␤-responsive gene expression (2-4).The TGF-␤ signaling pathway is inactivated in many tumors. Loss of negative growth regulation by TGF-␤ affords cells a selective growth advantage associated with decreased dependence of exogenous growth factor and increased tumorigenicity. Frequently, inhibition of TGF-␤ signaling occurs by either abolition of the function of a common mediator, Smad4, or interference with T␤RII function (5, 6). Smad4 and T␤RII are tumor suppressor gene...

Deficiency in either the interferon consensus sequence binding protein (ICSBP) or neurofibromin 1 (Nf1) increases the proliferative response of myeloid progenitor cell to hematopoietic cytokines. Consistent with this, we previously demonstrated that ICSBP activates transcription of the gene encoding Nf1 (the NF1 gene). In the studies presented here, we determine that ICSBP tyrosine phosphorylation is necessary for the activation of NF1 transcription. Since ICSBP is tyrosine phosphorylated in response to hematopoietic cytokines, these studies identify a novel pathway by which cytokine-induced posttranslational modification of ICSBP results in NF1 transcription. Nf1 subsequently inactivates cytokine-activated Ras, thereby creating a negative feedback mechanism for cytokine-induced proliferation. In these studies, we also determine that ICSBP is a substrate for SHP2 protein tyrosine phosphatase (SHP2-PTP). We find that wild-type SHP2-PTP dephosphorylates ICSBP only in undifferentiated myeloid cells. In contrast, a leukemia-associated, constitutively activated mutant form of SHP2-PTP dephosphorylates ICSBP in both myeloid progenitors and differentiating myeloid cells. Activated SHP2-PTP mutants thereby inhibit ICSBP-dependent NF1 transcription, impairing this negative feedback mechanism on cytokine-activated Ras. Therefore, these studies suggest that leukemia-associated ICSBP deficiency cooperates with leukemia-associated activating mutants of SHP2-PTP to contribute to the proliferative phenotype in myeloid malignancies.