birinapant and Leukemia--Myeloid--Acute

While oncogenes promote tumorigenesis, they also induce deleterious cellular stresses, such as apoptosis, that cancer cells must combat by coopting adaptive responses. Whether tumor suppressor gene haploinsufficiency leads to such phenomena and their mechanistic basis is unclear. Here, we demonstrate that elevated levels of the anti-apoptotic factor, CASP8 and FADD-like apoptosis regulator (CFLAR), promotes apoptosis evasion in acute myeloid leukemia (AML) cells haploinsufficient for the cut-like homeobox 1 (CUX1) transcription factor, whose loss is associated with dismal clinical prognosis. Genome-wide CRISPR/Cas9 screening identifies CFLAR as a selective, acquired vulnerability in CUX1-deficient AML, which can be mimicked therapeutically using inhibitor of apoptosis (IAP) antagonists in murine and human AML cells. Mechanistically, CUX1 deficiency directly alleviates CUX1 repression of the CFLAR promoter to drive CFLAR expression and leukemia survival. These data establish how haploinsufficiency of a tumor suppressor is sufficient to induce advantageous anti-apoptosis cell survival pathways and concurrently nominate CFLAR as potential therapeutic target in these poor-prognosis leukemias.

Topics: Animals; Apoptosis; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatin Immunoprecipitation; Dipeptides; fms-Like Tyrosine Kinase 3; Gene Expression Regulation, Neoplastic; Gene Ontology; Genes, Tumor Suppressor; Haploinsufficiency; Hematopoietic Stem Cells; Homeodomain Proteins; Humans; Indoles; Kaplan-Meier Estimate; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Chronic; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Nuclear Proteins; Promoter Regions, Genetic; Protein Array Analysis; Repressor Proteins

Resistance to chemotherapy is a major problem in cancer treatment, and it is frequently associated with failure of tumor cells to undergo apoptosis. Birinapant, a clinical SMAC mimetic, had been designed to mimic the interaction between inhibitor of apoptosis proteins (IAPs) and SMAC/Diablo, thereby relieving IAP-mediated caspase inhibition and promoting apoptosis of cancer cells. We show that acute myeloid leukemia (AML) cells are sensitive to birinapant-induced death and that the clinical caspase inhibitor emricasan/IDN-6556 augments, rather than prevents, killing by birinapant. Deletion of caspase-8 sensitized AML to birinapant, whereas combined loss of caspase-8 and the necroptosis effector MLKL (mixed lineage kinase domain-like) prevented birinapant/IDN-6556-induced death, showing that inhibition of caspase-8 sensitizes AML cells to birinapant-induced necroptosis. However, loss of MLKL alone did not prevent a caspase-dependent birinapant/IDN-6556-induced death, implying that AML will be less likely to acquire resistance to this drug combination. A therapeutic breakthrough in AML has eluded researchers for decades. Demonstrated antileukemic efficacy and safety of the birinapant/emricasan combination in vivo suggest that induction of necroptosis warrants clinical investigation as a therapeutic opportunity in AML.

Topics: Apoptosis; Caspase 8; Caspase Inhibitors; Cell Line, Tumor; Dipeptides; Drug Resistance, Neoplasm; Drug Synergism; Humans; Indoles; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid, Acute; Necrosis; Pentanoic Acids; Tumor Cells, Cultured

Acute myeloid leukemia (AML) therapy has limited long-term efficacy because patients frequently develop disease relapse because of the inability of standard chemotherapeutic agents to target AML stem/progenitor cells. Here, we identify deregulated apoptotic components in AML stem/progenitor cells and investigate the individual and combinatorial effects of the novel inhibitor of apoptosis (IAP) protein antagonist and second mitochondrial-derived activator of caspases (SMAC) mimetic birinapant and demethylating epigenetic modulators.. Protein expression was measured by reversed-phase protein array in AML patient (n = 511) and normal (n = 21) samples and by western blot in drug-treated cells. The antileukemic activity of birinapant and demethylating agents was assessed in vitro and in an in vivo AML mouse xenograft model (n = 10 mice per group). All statistical tests were two-sided.. Compared with bulk AML cells, CD34(+)38(-) AML stem/progenitors expressed increased cIAP1 and caspase-8 levels and decreased SMAC levels (one-way analysis of variance followed by Tukey's multiple comparison test, P < .001). Birinapant induced death receptor-/caspase-8-mediated apoptosis in AML cells, including in AML stem/progenitor cells, but not in normal CD34(+) cells. Demethylating agents modulated extrinsic apoptosis pathway components and, when combined with birinapant, were highly synergistic in vitro (combination index < 1), and also more effective in vivo (P < .001, by Student t test, for the median survival of birinapant plus 5-azacytadine vs birinapant alone or vs controls).. cIAP1, SMAC, and caspase-8 appear to play a role in AML stem cell survival, and synergistic targeting of these cells with birinapant and demethylating agents shows potential utility in leukemia therapy.

Topics: Adult; Aged; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis Regulatory Proteins; Azacitidine; Blotting, Western; Caspase 8; Dipeptides; DNA Methylation; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; Indoles; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitochondrial Proteins; Neoplastic Stem Cells; Protein Array Analysis

Overexpression of the apoptosis repressor with caspase recruitment domain (ARC, also termed NOL3) protein predicts adverse outcome in patients with acute myeloid leukaemia (AML) and confers drug resistance to AML cells. The second mitochondrial-derived activator of caspases (SMAC, also termed DIABLO) mimetic, birinapant, promotes extrinsic apoptosis in AML cells. SMAC mimetics induce cleavage of cellular inhibitor of apoptosis (cIAP) proteins, leading to stabilization of the nuclear factor-κB (NF-κB)-inducing kinase (MAP3K14, also termed NIK) and activation of non-canonical NF-κB signalling. To enhance the therapeutic potential of SMAC mimetics in AML, we investigated the regulation and role of ARC in birinapant-induced apoptosis. We showed that birinapant increases ARC in AML and bone marrow-derived mesenchymal stromal cells (MSCs). Downregulation of MAP3K14 by siRNA decreased ARC levels and suppressed birinapant-induced ARC increase. Reverse-phase protein array analysis of 511 samples from newly diagnosed AML patients showed that BIRC2 (also termed cIAP1) and ARC were inversely correlated. Knockdown of ARC sensitized, while overexpression attenuated, birinapant-induced apoptosis. Furthermore, ARC knockdown in MSCs sensitized co-cultured AML cells to birinapant-induced apoptosis. Our data demonstrate that ARC is regulated via BIRC2/MAP3K14 signalling and its overexpression in AML or MSCs can function as a resistant factor to birinapant-induced leukaemia cell death, suggesting that strategies to inhibit ARC will improve the therapeutic potential of SMAC mimetics.

Topics: Aged; Antimetabolites, Antineoplastic; Apoptosis; Apoptosis Regulatory Proteins; Coculture Techniques; Dipeptides; Drug Design; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; Humans; Indoles; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid, Acute; MAP Kinase Signaling System; Mesenchymal Stem Cells; Middle Aged; Mitochondrial Proteins; Molecular Targeted Therapy; Muscle Proteins; NF-kappa B; NF-kappaB-Inducing Kinase; Protein Serine-Threonine Kinases; RNA Interference; RNA, Small Interfering; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Ubiquitin-Protein Ligases