Loss of Wilms tumor 1 protein is a marker for apoptosis in response to replicative stress in leukemic cells.

A remaining expression of the transcription factor Wilms tumor 1 (WT1) after cytotoxic chemotherapy indicates remaining leukemic clones in patients. We determined the regulation and relevance of WT1 in leukemic cells exposed to replicative stress and DNA damage. To induce these conditions, we used the clinically relevant chemotherapeutics hydroxyurea and doxorubicin. We additionally treated cells with the pro-apoptotic kinase inhibitor staurosporine. Our data show that these agents promote apoptosis to a variable extent in a panel of 12 leukemic cell lines and that caspases cleave WT1 during apoptosis. A chemical inhibition of caspases as well as an overexpression of mitochondrial, anti-apoptotic BCL2 family proteins significantly reduces the processing of WT1 and cell death in hydroxyurea-sensitive acute promyelocytic leukemia cells. Although the reduction of WT1 correlates with the pharmacological efficiency of chemotherapeutics in various leukemic cells, the elimination of WT1 by different strategies of RNA interference (RNAi) does not lead to changes in the cell cycle of chronic myeloid leukemia K562 cells. RNAi against WT1 does also not increase the extent of apoptosis and the accumulation of γH2AX in K562 cells exposed to hydroxyurea. Likewise, a targeted genetic depletion of WT1 in primary oviduct cells does not increase the levels of γH2AX. Our findings position WT1 as a downstream target of the apoptotic process that occurs in response to cytotoxic forms of replicative stress and DNA damage.

A phosphorylation-acetylation switch regulates STAT1 signaling.

Cytokines such as interferons (IFNs) activate signal transducers and activators of transcription (STATs) via phosphorylation. Histone deacetylases (HDACs) and the histone acetyltransferase (HAT) CBP dynamically regulate STAT1 acetylation. Here we show that acetylation of STAT1 counteracts IFN-induced STAT1 phosphorylation, nuclear translocation, DNA binding, and target gene expression. Biochemical and genetic experiments altering the HAT/HDAC activity ratio and STAT1 mutants reveal that a phospho-acetyl switch regulates STAT1 signaling via CBP, HDAC3, and the T-cell protein tyrosine phosphatase (TCP45). Strikingly, inhibition of STAT1 signaling via CBP-mediated acetylation is distinct from the functions of this HAT in transcriptional activation. STAT1 acetylation induces binding of TCP45, which catalyzes dephosphorylation and latency of STAT1. Our results provide a deeper understanding of the modulation of STAT1 activity. These findings reveal a new layer of physiologically relevant STAT1 regulation and suggest that a previously unidentified balance between phosphorylation and acetylation affects cytokine signaling.

Mechanism for ubiquitylation of the leukemia fusion proteins AML1-ETO and PML-RARalpha.

The chromosomal translocation products AML1-ETO and PML-RARalpha contribute to the pathogenesis of leukemias. Here, we demonstrate that both AML1-ETO and PML-RARalpha are degraded by the ubiquitin-proteasome system and that their turnover critically depends on the E2-conjugase UbcH8 and the E3-ligase SIAH-1. Contrary to its role in HDAC2 degradation, the E3-ligase RLIM does not target AML1-ETO and PML-RARalpha for ubiquitin-dependent elimination. RLIM rather is a substrate of SIAH-1, which indicates that these E3-ligases operate in a hierarchical order. Remarkably, proteasomal degradation of leukemia fusion proteins, in addition to the block of histone deacetylase (HDAC) enzymatic activity is a consequence of HDAC-inhibitor treatment. The former requires the induction of UbcH8 expression and each of these processes might be beneficial for leukemia treatment. Our observations shed light on the mechanism determining the interplay between E2-conjugases, E3-ligases, and their substrates and suggest a strategy for utilizing the ubiquitylation machinery in a therapeutic setting.

HSP90 is necessary for the ACK1-dependent phosphorylation of STAT1 and STAT3.

Signal transducers and activators of transcription (STATs) are latent, cytoplasmic transcription factors. Janus kinases (JAKs) and activated CDC42-associated kinase-1 (ACK1/TNK2) catalyse the phosphorylation of STAT1 and the expression of its target genes. Here we demonstrate that catalytically active ACK1 promotes the phosphorylation and nuclear accumulation of STAT1 in transformed kidney cells. These processes are associated with STAT1-dependent gene expression and an interaction between endogenous STAT1 and ACK1. Moreover, the E3 ubiquitin ligase seven-in-absentia homolog-2 (SIAH2), which targets ACK1 through valine-909 for proteasomal degradation, attenuates the ACK1-STAT1 signalling node. We further show that ACK1 promotes the phosphorylation and nuclear accumulation of STAT3 in cultured cells and that the levels of ACK1 correlate positively with the levels of tyrosine phosphorylated STAT3 in primary lung adenocarcinoma (ADC) cells. Global analysis of ACK1 interaction partners validated the interaction of ACK1 with heat shock protein 90 (HSP90α/ß). Inhibition of this chaperone with the novel drug Onalespib (AT13387) demonstrates that HSP90 is an upstream regulator of the ACK1-dependent phosphorylation of STAT1 and STAT3. In addition to these molecular insights, our data offer a pharmacological strategy to control the ACK1-STAT signalling axis.

A combination of a ribonucleotide reductase inhibitor and histone deacetylase inhibitors downregulates EGFR and triggers BIM-dependent apoptosis in head and neck cancer.

Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common malignant neoplasm and more than 50% of patients succumb to this disease. HNSCCs are characterized by therapy resistance, which relies on the overexpression of anti-apoptotic proteins and on the aberrant regulation of the epidermal growth factor receptor (EGFR). As inherent and acquired resistance to therapy counteracts improvement of long-term survival, novel multi-targeting strategies triggering cancer cell death are urgently required. We investigated how induction of replicational stress by the ribonucleotide reductase inhibitor hydroxyurea (HU) combined with histone deacetylase inhibitors (HDACi) exerts anti-tumor activity. We treated HNSCC cell lines and freshly isolated tumor cells with HDACi, such as the clinically approved anti-epileptic drug valproic acid (VPA), in combination with HU. Our data demonstrate that at clinically achievable levels VPA/HU combinations efficiently block proliferation as well as clonogenic survival, and trigger apoptosis of HNSCC cells. In the presence of VPA/HU, such tumor cells increase expression of the pro-apoptotic BCL-2 family protein BIM, independent of wild-type p53 signaling and in the absence of increased expression of the p53 targets PUMA and BAX. The pro-apoptotic activity of BIM in HNSCCs was found critical for tumor cell death; ectopic overexpression of BIM induced HNSCC apoptosis and RNAi-mediated depletion of BIM protected HNSCC cells from VPA/HU. Also, significantly elevated BIM levels (p less than 0.01) were detectable in the apoptotic tumor centers versus proliferating tumor margins in HNSCC patients (n=31), underlining BIM's clinical relevance. Importantly, VPA/HU treatment additionally reduces expression and cell surface localization of EGFR. Accordingly, in a xenograft mouse model, VPA/HU efficiently blocked tumor growth (P less than 0.001) correlating with BIM induction and EGFR downregulation. We provide a molecular rationale for the potent anti-cancer activities of this drug combination. Our data suggest its exploitation as a potential strategy for the treatment of HNSCC and other tumor entities characterized by therapy resistance linked to dysregulated EGFR activation.