Induction of necroptosis has emerged as an alternative approach to trigger programmed cell death, in particular in apoptosis-resistant cancer cells. Recent evidence suggests that kinase inhibitors targeting oncogenic B-RAF can also affect Receptor-interacting serine/threonine-protein kinase (RIP)1 and RIP3. Sorafenib, a multi-targeting kinase inhibitor with activity against B-RAF, is used for the treatment of acute leukemia. In the present study, we therefore investigated whether Sorafenib interferes with therapeutic induction of necroptosis in acute leukemia. Here, we report that Sorafenib inhibits necroptotic signaling and cell death in two models of necroptosis in acute leukemia. Sorafenib significantly reduces Second mitochondria-derived activator of caspases (Smac) mimetic-induced necroptosis in apoptosis-resistant acute myeloid leukemia (AML) cells as well as Smac mimetic/Tumor Necrosis Factor (TNF)α-induced necroptosis in FADD-deficient acute lymphoblastic leukemia (ALL) cells. Sub-to low micromolar concentrations of Sorafenib corresponding to its plasma levels reported in cancer patients are sufficient to inhibit necroptosis, emphasizing the clinical relevance of our findings. Furthermore, Sorafenib blocks Smac mimetic-mediated phosphorylation of mixed-lineage kinase domain-like protein (MLKL) that marks its activation, indicating that Sorafenib targets components upstream of MLKL such as RIP1 and RIP3. Intriguingly, Sorafenib reduces the Smac mimetic/TNFα-stimulated interaction of RIP1 with RIP3 and MLKL, demonstrating that it interferes with the assembly of the necrosome complex. Importantly, Sorafenib significantly protects primary, patient-derived AML blasts from Smac mimetic-induced necroptosis. By demonstrating that Sorafenib limits the anti-leukemic activity of necroptosis-inducing drugs in acute leukemia cells, our study has important implications for the use of Sorafenib in the treatment of acute leukemia.

Redox mechanisms play an important role in the control of various signaling pathways. Here, we report that Second mitochondrial activator of caspases (Smac) mimetic-induced cell death is regulated by redox signaling. We show that RSL3, a glutathione (GSH) peroxidase (GPX) 4 inhibitor, or Erastin, an inhibitor of the cystine/glutamate antiporter, cooperate with the Smac mimetic BV6 to induce reactive oxygen species (ROS)-dependent cell death in acute lymphoblastic leukemia (ALL) cells. Addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to rescue ROS-induced cell death, demonstrating that RSL3/BV6- or Erastin/BV6-induced cell death occurs in a caspase-independent manner. Interestingly, the iron chelator Deferoxamine (DFO) significantly inhibits RSL3/BV6-induced cell death, whereas it is unable to rescue cell death by Erastin/BV6, showing that RSL3/BV6-, but not Erastin/BV6-mediated cell death depends on iron. ROS production is required for both RSL3/BV6- and Erastin/BV6-induced cell death, since the ROS scavenger α-tocopherol (α-Toc) rescues RSL3/BV6- and Erastin/BV6-induced cell death. By comparison, genetic or pharmacological inhibition of lipid peroxidation by GPX4 overexpression or ferrostatin (Fer)-1 significantly decreases RSL3/BV6-, but not Erastin/BV6-induced cell death, despite inhibition of lipid peroxidation upon exposure to RSL3/BV6 or Erastin/BV6. Of note, inhibition of lipid peroxidation by Fer-1 protects from RSL3/BV6-, but not from Erastin/BV6-stimulated ROS production, indicating that other forms of ROS besides lipophilic ROS occur during Erastin/BV6-induced cell death. Taken together, RSL3/BV6 and Erastin/BV6 differentially regulate redox signaling and cell death in ALL cells. While RSL3/BV6 cotreatment induces ferroptotic cell death, Erastin/BV6 stimulates oxidative cell death independently of iron. These findings have important implications for the therapeutic targeting of redox signaling to enhance Smac mimetic-induced cell death in ALL.

Necroptosis is a form of programmed cell death that critically depends on RIP3 and MLKL. However, the contribution of mitochondria to necroptosis is still poorly understood. In the present study, we discovered that mitochondrial perturbations play a critical role in Smac mimetic/Dexamethasone (Dexa)-induced necroptosis independently of death receptor ligands. We demonstrate that the Smac mimetic BV6 and Dexa cooperate to trigger necroptotic cell death in acute lymphoblastic leukemia (ALL) cells that are deficient in caspase activation due to absent caspase-8 expression or pharmacological inhibition by the caspase inhibitor zVAD.fmk, since genetic silencing or pharmacological inhibition of RIP3 or MLKL significantly rescue BV6/Dexa-induced necroptosis. In addition, RIP3 or MLKL knockout mouse embryonic fibroblasts (MEFs) are protected from BV6/Dexa/zVAD.fmk-induced cell death. In contrast, antagonistic antibodies against the death receptor ligands TNFα, TRAIL or CD95 ligand fail to rescue BV6/Dexa-triggered cell death. Kinetic studies revealed that prior to cell death BV6/Dexa treatment causes hyperpolarization of the mitochondrial membrane potential (MMP) followed by loss of MMP, reactive oxygen species (ROS) production, Bak activation and disruption of mitochondrial respiration. Importantly, knockdown of Bak significantly reduces BV6/Dexa-induced loss of MMP and delays cell death, but not ROS production, whereas ROS scavengers attenuate Bak activation, indicating that ROS production occurs upstream of BV6/Dexa-mediated Bak activation. Consistently, BV6/Dexa treatment causes oxidative thiol modifications of Bak protein. Intriguingly, knockdown or knockout of RIP3 or MLKL protect ALL cells or MEFs from BV6/Dexa-induced ROS production, Bak activation, drop of MMP and disruption of mitochondrial respiration, demonstrating that these mitochondrial events depend on RIP3 and MLKL. Thus, mitochondria might serve as an amplification step in BV6/Dexa-induced necroptosis. These findings provide new insights into the role of mitochondrial dysfunctions during necroptosis and have important implications for the development of novel treatment approaches to overcome apoptosis resistance in ALL.