TNF signaling inactivation sensitizes AML cells to NF-kB inhibition but protects healthy hematopoietic stem progenitor cells from this treatment.

Mutations and inactivation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) are observed in 15%-25% of cases of human T cell acute lymphoblastic leukemia (T-ALL). Pten deletion induces myeloproliferative disorders (MPDs), acute myeloid leukemia (AML), and/or T-ALL in mice. Previous studies attributed Pten-loss-related hematopoietic defects and leukemogenesis to excessive activation of phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling. Although inhibition of this signal dramatically suppresses the growth of PTEN-null T-ALL cells in vitro, treatment with inhibitors of this pathway does not cause a complete remission in vivo. Here, we report that focal adhesion kinase (Fak), a protein substrate of Pten, also contributes to T-ALL development in Pten-null mice. Inactivation of the FAK signaling pathway by either genetic or pharmacologic methods significantly sensitizes both murine and human PTEN-null T-ALL cells to PI3K/AKT/mTOR inhibition when cultured in vitro on feeder layer cells or a matrix and in vivo.

Objective Reduced blood flow and/or tissue oxygen tension conditions result from thrombotic and vascular diseases such as myocardial infarction, stroke, and peripheral vascular disease. It is largely assumed that while platelet activation is increased by an acute vascular event, chronic vascular inflammation, and/or ischemia, the platelet activation pathways and responses are not themselves changed by the disease process. We therefore sought to determine whether the platelet phenotype is altered by hypoxic and ischemic conditions. Approach and Results In a cohort of patients with metabolic and peripheral artery disease (PAD), platelet activity was enhanced and/or inhibition with oral anti-platelet agents was impaired compared to platelets from control subjects, suggesting a difference in platelet phenotype caused by disease. Isolated murine and human platelets exposed to reduced oxygen (hypoxia chamber, 5% O2) had increased expression of some proteins that augment platelet activation compared to platelets in normoxic conditions (21% O2). Using a murine model of critical limb ischemia (CLI), platelet activity was increased even two weeks post-surgery compared to sham surgery mice. This effect was partly inhibited in platelet specific Extracellular Regulated Protein Kinase 5 (ERK5) knockout mice. Conclusions These findings suggest that ischemic disease changes the platelet phenotype and alters platelet agonist responses due to changes in the expression of signal transduction pathway proteins. Platelet phenotype and function should therefore be better characterized in ischemic and hypoxic diseases to understand the benefits and limitations of anti-platelet therapy.

Tumor necrosis factor-α (TNF)-induced RIP1/RIP3-mediated necroptosis has been proposed to be an alternative strategy for treating apoptosis-resistant leukemia. However, we found that most acute myeloid leukemia (AML) cells, especially M4 and M5 subtypes, produce TNF and show basal level activation of RIP1/RIP3/MLKL signaling, yet do not undergo necroptosis. TNF, through RIP1/RIP3 signaling, prevents degradation of SOCS1, a key negative regulator of interferon-γ (IFN-γ) signaling. Using both pharmacologic and genetic assays, we show here that inactivation of RIP1/RIP3 resulted in reduction of SOCS1 protein levels and partial differentiation of AML cells. AML cells with inactivated RIP1/RIP3 signaling show increased sensitivity to IFN-γ-induced differentiation. RIP1/RIP3 inactivation combined with IFN-γ treatment significantly attenuated the clonogenic capacity of both primary AML cells and AML cell lines. This combination treatment also compromised the leukemogenic ability of murine AML cells in vivo. Our studies suggest that inhibition of RIP1/RIP3-mediated necroptotic signaling might be a novel strategy for the treatment of AML when combined with other differentiation inducers.

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