RIP1 is required for IAP inhibitor-mediated sensitization of childhood acute leukemia cells to chemotherapy-induced apoptosis

Abstract

Evasion of apoptosis may contribute to poor treatment response in pediatric acute lymphoblastic leukemia (ALL), calling for novel treatment strategies. Here, we report that inhibitors of apoptosis (IAPs) at subtoxic concentrations cooperate with various anticancer drugs (that is, AraC, Gemcitabine, Cyclophosphamide, Doxorubicin, Etoposide, Vincristine and Taxol) to induce apoptosis in ALL cells in a synergistic manner as calculated by combination index and to reduce long-term clonogenic survival. Importantly, we identify RIP1 as a critical regulator of this synergism of IAP inhibitors and AraC that mediates the formation of a RIP1/FADD/caspase-8 complex via an autocrine/paracrine loop of tumor necrosis factor-a (TNFa). Knockdown of RIP1 abolishes formation of this complex and subsequent activation of caspase-8 and -3, mitochondrial perturbations and apoptosis. Similarly, inhibition of RIP1 kinase activity by Necrostatin-1 or blockage of TNFa by Enbrel inhibits IAP inhibitor-and AraC-triggered interaction of RIP1, FADD and caspase-8 and apoptosis. In contrast to malignant cells, IAP inhibitors and AraC at equimolar concentrations are non-toxic to normal peripheral blood lymphocytes or mesenchymal stromal cells. Thus, our findings provide first evidence that IAP inhibitors present a promising strategy to prime childhood ALL cells for chemotherapy-induced apoptosis in a RIP1-dependent manner. These data have important implications for developing apoptosis-targeted therapies in childhood leukemia.

Smac mimetic and glucocorticoids synergize to induce apoptosis in childhood ALL by promoting ripoptosome assembly

“…Previously, we reported that small-molecule inhibitors of IAP proteins such as Smac mimetics act in concert with other cytotoxic stimuli, for example death receptor ligands such as TRAIL and CD95 ligand, chemotherapeutic drugs, or irradiation, in ALL or other cancer entities. 24,25,38,[40][41][42][43] Second, we identify a novel underlying molecular mechanism of this synergistic interaction by showing that BV6 and dexamethasone act in concert to trigger proteasomal degradation of IAP proteins, which in turn promotes the assembly of the ripoptosome, a cytosolic cell death complex ( Figure 6). Several lines of evidence support this conclusion, as follows:…”

“…Leukemia cells, human mesenchymal cells (MSCs), human G-CSF mobilized CD34 1 (immature) hematopoietic cells, monocytes, and dendritic cells were cultured as described previously 23,25 or as described in supplemental Methods BLOOD, 10 JULY 2014 x VOLUME 124, NUMBER 2 SYNERGISTIC LETHALITY OF Smac AND GLUCOCORTICOIDS 241…”

“…by guest www.bloodjournal.org From potential and Bax/Bak activation were examined as described, 28 reactive oxygen species (ROS) production by flow cytometry using 1 mM CellROX or 5 mM MitoSox (Invitrogen). Western blotting 27 and immunoprecipitation 25 were done as described.…”

“…22 We previously reported that small-molecule IAP inhibitors enhance death receptoror chemotherapy-induced apoptosis in childhood leukemia. [23][24][25] However, the question of whether or not targeting IAP proteins represents a suitable strategy to bypass GC resistance in ALL has not yet been answered. Therefore, in the present study we investigated the question of whether or not neutralization of XIAP, cIAP1, and cIAP2 by the small-molecule Smac mimetic BV6 19 can enhance the antileukemic activity of GC against childhood ALL.…”

RIPK1 and RIPK3 - emerging targets in cancer?

“…Activation of RIPK1/RIPK3-dependent apoptosis or necroptosis typically requires additional modifiers, such as inhibition of cIAPs for activation of RIPK1 kinase-dependent apoptosis (73,74) and/or inhibition of caspase-8 for RIPK1/RIPK3 kinasedependent necroptosis (64,66,75,76). Consequently, Moriwaki et al examined cell death in response to a panel of commonly used chemotherapeutic agents and did not observe any changes in cell death upon knockdown of either RIPK1 or RIPK3 in RIPK3-expressing cells or upon overexpression of RIPK3 in cells that lost RIPK3 expression(37), suggesting that RIPK1/RIPK3 may not be generally involved in chemotherapy-induced cell death.…”