p65/RelA NF-κB fragments generated by RIPK3 activity regulate tumorigenicity, cell metabolism, and stemness characteristics.

Receptor-interacting protein kinase 3 (RIPK3) can induce necroptosis, apoptosis, or cell proliferation and is silenced in several hematological malignancies. We previously reported that RIPK3 activity independent of its kinase domain induces caspase-mediated p65/RelA cleavage, resulting in N-terminal 1-361 and C-terminal 362-549 fragments. We show here that a noncleavable p65/RelA D361E mutant expressed in DA1-3b leukemia cells decreases mouse survival times and that coexpression of p65/RelA fragments increases the tumorigenicity of B16F1 melanoma cells. This aggressiveness in vivo did not correlate with NF-κB activity measured in vitro. The fragments and p65/RelA D361E mutant induced different expression profiles in DA1-3b and B16F1 cells. Stemness markers were affected: p65/RelA D361E increased ALDH activity in DA1-3b cells, and fragment expression increased melanoma sphere formation in B16/F1 cells. p65/RelA fragments and the D361E noncleavable mutant decreased oxidative or glycolytic cell metabolism, with differences observed between models. Thus, p65/RelA cleavage initiated by kinase-independent RIPK3 activity in cancer cells is not neutral and induces pleiotropic effects in vitro and in vivo that may vary across tumor types.

Identification of a novel synergistic induction of cell death by Smac mimetic and HDAC inhibitors in acute myeloid leukemia cells.

Inhibitor of Apoptosis (IAP) proteins are expressed at high levels in acute myeloid leukemia (AML) and contribute to resistance to programmed cell death. Here, we report that inhibition of IAP proteins by the small-molecule Smac mimetic BV6 acts together with histone deacetylase (HDAC) inhibitors (HDACIs) such as MS275 or SAHA to trigger cell death in AML cell lines in a synergistic manner, as underscored by calculation of combination index (CI). Also, BV6 and HDACIs cooperate to trigger DNA fragmentation, a marker of apoptotic cell death, and to suppress long-term clonogenic survival of AML cells. In contrast, equimolar concentrations of BV6 and MS275 or SAHA do not synergize to elicit cell death in normal peripheral blood lymphocytes (PBLs), emphasizing some tumor cell selectivity of this combination treatment. Addition of the tumor necrosis factor (TNF)α-blocking antibody Enbrel significantly reduces BV6/MS275-induced cell death in the majority of AML cell lines, indicating that autocrine/paracrine TNFα signaling contributes to cell death. Remarkably, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to rescue MV4-11, Molm13 and OCI-AML3 cells and even enhances BV6/MS275-mediated cell death, whereas zVAD.fmk reduces BV6/MS275-induced cell death in NB4 cells. Annexin-V/propidium iodide (PI) double staining reveals that BV6/MS275 cotreatment predominately increases the percentage of double-positive cells. Of note, the Receptor-Interacting Protein (RIP)1 inhibitor necrostatin-1 (Nec-1) or the Mixed Lineage Kinase Domain-Like protein (MLKL) inhibitor necrosulfonamide (NSA) significantly reduce BV6/MS275-induced cell death in the presence of zVAD.fmk, suggesting that BV6/MS275 cotreatment triggers necroptosis when caspases are inhibited. Thus, BV6 acts in concert with HDACIs to induce cell death in AML cells and can bypass apoptosis resistance, at least in several AML cell lines, by engaging necroptosis as an alternative route of regulated cell death. The identification of a novel synergism of BV6 and HDACIs has important implications for the development of new treatment strategies for AML.