Targeted PI3K/AKT-hyperactivation induces cell death in chronic lymphocytic leukemia.

Current therapeutic approaches for chronic lymphocytic leukemia (CLL) focus on the suppression of oncogenic kinase signaling. Here, we test the hypothesis that targeted hyperactivation of the phosphatidylinositol-3-phosphate/AKT (PI3K/AKT)-signaling pathway may be leveraged to trigger CLL cell death. Though counterintuitive, our data show that genetic hyperactivation of PI3K/AKT-signaling or blocking the activity of the inhibitory phosphatase SH2-containing-inositol-5'-phosphatase-1 (SHIP1) induces acute cell death in CLL cells. Our mechanistic studies reveal that increased AKT activity upon inhibition of SHIP1 leads to increased mitochondrial respiration and causes excessive accumulation of reactive oxygen species (ROS), resulting in cell death in CLL with immunogenic features. Our results demonstrate that CLL cells critically depend on mechanisms to fine-tune PI3K/AKT activity, allowing sustained proliferation and survival but avoid ROS-induced cell death and suggest transient SHIP1-inhibition as an unexpectedly promising concept for CLL therapy.

AKT-dependent NOTCH3 activation drives tumor progression in a model of mesenchymal colorectal cancer.

Recently, a transcriptome-based consensus molecular subtype (CMS) classification of colorectal cancer (CRC) has been established, which may ultimately help to individualize CRC therapy. However, the lack of animal models that faithfully recapitulate the different molecular subtypes impedes adequate preclinical testing of stratified therapeutic concepts. Here, we demonstrate that constitutive AKT activation in intestinal epithelial cells markedly enhances tumor invasion and metastasis in Trp53ΔIEC mice (Trp53ΔIECAktE17K) upon challenge with the carcinogen azoxymethane. Gene-expression profiling indicates that Trp53ΔIECAktE17K tumors resemble the human mesenchymal colorectal cancer subtype (CMS4), which is characterized by the poorest survival rate among the four CMSs. Trp53ΔIECAktE17K tumor cells are characterized by Notch3 up-regulation, and treatment of Trp53ΔIECAktE17K mice with a NOTCH3-inhibiting antibody reduces invasion and metastasis. In CRC patients, NOTCH3 expression correlates positively with tumor grading and the presence of lymph node as well as distant metastases and is specifically up-regulated in CMS4 tumors. Therefore, we suggest NOTCH3 as a putative target for advanced CMS4 CRC patients.

Detection of NF-κB pathway activation in T helper cells.

Upon activation of CD4+ T helper cells the transcription factor complex NF-κB becomes activated and its subunits are able to translocate to the nucleus. There, the dimerized proteins regulate the transcription of for example cytokine and pro-survival genes. This process is of central importance for a proper function of T helper cells as its loss causes severe immunodeficiency, whereas its deregulation can contribute to lymphomagenesis or autoimmunity. In this protocol we describe four methods to investigate NF-κB activation in T helper cells by testing (1) the assembly of the Carma1-Malt1-Bcl10 complex by co-immunoprecipitation, (2) the activation of the IKK complex and the degradation of IκBα by western blot analysis, (3) the degradation of IκBα by intracellular flow cytometry, and (4) the nuclear translocation and DNA binding activity of NF-κB by nonradioactive electrophoretic mobility shift assay (EMSA).

Protein kinase c-ß-dependent activation of NF-κB in stromal cells is indispensable for the survival of chronic lymphocytic leukemia B cells in vivo.

Tumor cell survival critically depends on heterotypic communication with benign cells in the microenvironment. Here, we describe a survival signaling pathway activated in stromal cells by contact to B cells from patients with chronic lymphocytic leukemia (CLL). The expression of protein kinase C (PKC)-ßII and the subsequent activation of NF-κB in bone marrow stromal cells are prerequisites to support the survival of malignant B cells. PKC-ß knockout mice are insusceptible to CLL transplantations, underscoring the in vivo significance of the PKC-ßII-NF-κB signaling pathway in the tumor microenvironment. Upregulated stromal PKC-ßII in biopsies from patients with CLL, acute lymphoblastic leukemia, and mantle cell lymphoma suggests that this pathway may commonly be activated in a variety of hematological malignancies.