Therapeutic targeting of necroptosis by Smac mimetic bypasses apoptosis resistance in acute myeloid leukemia cells

Abstract

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.

Sorafenib tosylate inhibits directly necrosome complex formation and protects in mouse models of inflammation and tissue injury

“…Since Sorafenib is used to treat AML, 27 we also included two AML cell lines to investigate whether it alters the sensitivity of these cells to necroptotic stimuli (BV6 in the presence of zVAD-fmk). 34 Sorafenib significantly reduced BV6/zVAD-fmk-induced necroptosis of AML cells in a dose-dependent manner (Figures 2e and f). Similar to the L929sAhFas cell line (Figure 1c), the human AML cells underwent Sorafenib-induced cell death (EC50 of 19.6  μ M for MV4-11 cells) (Supplementary Figures 4C and D), while they were protected against BV6+zVAD (IC50 Molm13 of⩽0.03  μ M) (Figures 2e and f).…”

“…Smac mimetic BV6 34 instead of TAK1i was used as a sensitizer for testing of mouse embryonic fibroblasts (MEF) and human cell lines. Dose-response curves and IC50 values for the potency of Sorafenib to inhibit cell death, were determined and compared with Nec-1s, as a reference compound.…”

Targeting RIPK1 in AML cells carrying FLT3‐ITD

“…In particular, the study from Kasper et al revealed significant upregulation of Mcl‐1 and showed that silencing of Mcl‐1 sensitized 32D‐FLT3‐ITD cells effectively toward cell death. Next to antiapoptotic signaling, also key proteins of the DR networks like RIPK3 and caspase‐8 have been suggested before to be important for the sensitization of AML cells carrying an FLT3‐ITD mutation . In particular, RIPK3 was reported to be suppressed in AML cells with an FLT3‐ITD mutation .…”

“…Moreover, several studies reported an overexpression of IAP proteins in AML cells which is associated with poor prognosis . Furthermore, a number of recent reports pointed out the importance of using SMAC mimetics in AML cells to promote induction of cell death pathways …”

Necroinflammation emerges as a key regulator of hematopoiesis in health and disease

“…In cell line models, the data supporting a relevant role of IAP antagonists against hematopoietic cancers was relatively positive. Several reports show that the IAP antagonist BV6 promoted elimination of ALL and AML cells as single agent or in combination with additional chemotherapeutics such as cytarabine or epigenetic modifiers (i.e., demethylating agents, histone deacetylase inhibitors), ionizing radiation, TRAIL, IFNα, and others [136][137][138][139][140][141][142]. The therapeutic benefit of these IAP antagonists was placed on the possibility to induce cell death also in cell lines that were refractory to chemo-or radiotherapy.…”