Apoptosis resistance commonly occurs in cancers, preventing activation of Caspase family cell death proteases. XIAP is an endogenous inhibitor of Caspases overexpressed in many cancers. We developed an enzyme derepression assay, based on overcoming XIAP-mediated suppression of Caspase-3, and screened mixture-based combinatorial chemical libraries for compounds that reversed XIAP-mediated inhibition of Caspase-3, identifying a class of polyphenylureas with XIAP-inhibitory activity. These compounds, but not inactive structural analogs, stimulated increases in Caspase activity, directly induced apoptosis of many types of tumor cell lines in culture, and sensitized cancer cells to chemotherapeutic drugs. Active compounds also suppressed growth of established tumors in xenograft models in mice, while displaying little toxicity to normal tissues. These findings validate IAPs as targets for cancer drug discovery.

Bax inhibitor-1 (BI-1) is an evolutionarily conserved endoplasmic reticulum (ER) protein that suppresses cell death in both animal and plant cells. We characterized mice in which the bi-1 gene was ablated. Cells from BI-1-deficient mice, including fibroblasts, hepatocytes, and neurons, display selective hypersensitivity to apoptosis induced by ER stress agents (thapsigargin, tunicamycin, brefeldin A), but not to stimulators of mitochondrial or TNF/Fas-death receptor apoptosis pathways. Conversely, BI-1 overexpression protects against apoptosis induced by ER stress. BI-1-mediated protection from apoptosis induced by ER stress correlated with inhibition of Bax activation and translocation to mitochondria, preservation of mitochondrial membrane potential, and suppression of caspase activation. BI-1 overexpression also reduces releasable Ca(2+) from the ER. In vivo, bi-1(-/-) mice exhibit increased sensitivity to tissue damage induced by stimuli that trigger ER stress, including stroke and tunicamycin injection. Thus, BI-1 regulates a cell death pathway important for cytopreservation during ER stress.

A simple immunopanel can divide breast cancers into biologic subtypes with strong prognostic effects. TAC significantly complements endocrine therapy in patients with luminal B subtype and, in the absence of targeted therapy, is effective in the triple-negative population.

Caspase-9 is critical for cytochrome c (cytoc)-dependent apoptosis and normal brain development. We determined that this apical protease in the cyto-c pathway for apoptosis resides inside mitochondria in several types of cells, including cardiomyocytes and many neurons. Caspase-9 is released from isolated mitochondria on treatment with Ca 2؉ or Bax, stimuli implicated in ischemic neuronal cell death that are known to induce cyto-c release from mitochondria. In neuronal cell culture models, apoptosis-inducing agents trigger translocation of caspase-9 from mitochondria to the nucleus, which is inhibitable by Bcl-2. Similarly, in an animal model of transient global cerebral ischemia, caspase-9 release from mitochondria and accumulation in nuclei was observed in hippocampal and other vulnerable neurons exhibiting early postischemic changes preceding apoptosis. Loss of mitochondrial barrier function during neuronal damage from ischemia or other insults therefore may play an important role in making certain caspases available to participate in apoptosis.

The patterns of expression of the bcl-2, bax, and bci-X genes were examined immunohistochemically in neurons of the adult rat brain before and after 10 min of global ischemia induced by transient cardiac arrest. High levels of the cell death promoting protein Bax and concomitant low levels of the apoptosis-blocking protein Bcl-2 were found in some populations of neurons that are particularly sensitive to cell death induced by transient global ischemia, such as the CA1 sector of the hippocampus and the Purkinje cells of the cerebellum. Moreover, within 0.5 to 3 hr after an ischemic episode, immunostaining for Bax was markedly increased within neurons with morphological features of degeneration in many regions of the brain. Use of a two-color staining method for simultaneous analysis of Bax protein and in situ detection of DNA-strand breaks revealed high levels of Bax immunoreactivity in many neurons undergoing apoptosis. Postischemic elevations in Bax protein levels in the hippocampus, cortex, and cerebellum were also demonstrated by immunoblotting. At early times after transient ischemia, regulation of Bcl-2 and Bcl-x protein levels varied among neuronal subpopulations, but from 3 hr on, those neurons with morphological evidence of degeneration uniformly contained reduced levels of Bci-2 and particularly Bci-X immunoreactivity. The findings suggest that differential expression of some members of the bcl-2 gene family may play an important role in determining the relative sensitivity of neuronal subpopulations to ischemia and that postischemic alterations in the expression of bax, bcl-2, and bcl-x may contribute to the delayed neuronal cell death that occurs during the repurfusion phase after a transient ischemic episode.

Proteins containing PAAD [pyrin, AIM (absent-in-melanoma), ASC [apoptosis-associated speck-like protein containing a CARD (caspase-recruitment domain)] and DD (death domain)-like] (PYRIN, DAPIN) domains are involved in innate immunity, regulating pathways leading to nuclear-factor-kappa B (NF-kappa B) and pro-caspase-1 activation. Many PAAD-family proteins have structures reminiscent of Nod-1, a putative intracellular sensor of lipopolysaccharide. Hereditary mutations in some of the PAAD-family genes are associated with auto-inflammatory diseases. Several of these proteins utilize the bipartite PAAD- and CARD-containing adapter protein ASC/TMS-1 (target of methylation-induced silencing) for linking to downstream signalling pathways. In the present paper, we describe characterization of human PAAD-only protein-1 (POP1)/ASC2, which is highly homologous with the PAAD domain of ASC, and which probably originated by gene duplication on chromosome 16. We demonstrate that POP1/ASC2 associates with ASC via PAAD-PAAD interactions and modulates NF-kappa B and pro-caspase-1 regulation by this adapter protein. In gene transfer experiments, POP1/ASC2 suppressed cytokine-mediated NF-kappa B activation similar to other PAAD-family proteins previously tested. Immunohistochemical studies showed expression of POP1/ASC2 predominantly in macrophages and granulocytes. We propose that POP1/ASC2 functions as a modulator of multidomain PAAD-containing proteins involved in NF-kappa B and pro-caspase-1 activation and innate immunity.

Summary Leukemia stem cells (LSC) play a pivotal role in chronic myeloid leukemia (CML) tyrosine kinase inhibitor (TKI) resistance and progression to blast crisis (BC), in part, through alternative splicing of self-renewal and survival genes. To elucidate splice isoform regulators of human BC LSC maintenance, we performed whole transcriptome RNA sequencing; splice isoform-specific qRT-PCR, nanoproteomics, stromal co-culture and BC LSC xenotransplantation analyses. Cumulatively, these studies show that alternative splicing of multiple pro-survival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSC that are quiescent in the marrow niche and contribute to therapeutic resistance. Notably, a novel pan-BCL2 inhibitor, sabutoclax, renders marrow niche-resident BC LSC sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice isoform expression in BC LSC maintenance and suggest that combinatorial inhibition of pro-survival BCL2 family proteins and BCR-ABL may eliminate dormant LSC and obviate resistance.

Ischemia-reperfusion (IR) injury induces endoplasmic reticulum (ER) stress and cell death. Bax Inhibitor-1 (BI-1) is an evolutionarily conserved ER protein that suppresses cell death and that is abundantly expressed in both liver and kidney. We explored the role of BI-1 in protection from ER stress and IR injury by using bi-1 knockout mice, employing models of transient hepatic or renal artery occlusion. Compared to wild-type bi-1 mice, bi-1 knockout mice subjected to hepatic IR injury exhibited these characteristics: (i) increased histological injury; (ii) increased serum transaminases, indicative of more hepatocyte death; (iii) increased percentages of TUNEL-positive hepatocytes; (iv) greater elevations in caspase activity; and (v) more activation of ER stress proteins inositol-requiring enzyme 1 and activating transcription factor 6 and greater increases in expression of ER stress proteins C/EBP homologous protein and spliced XBP-1 protein. Moreover, hepatic IR injury induced elevations in bi-1 mRNA in wild-type liver, suggesting a need for bi-1 gene induction to limit tissue injury. Similar sensitization of kidney to ER stress and IR injury was observed in bi-1(-/-) mice. We conclude that bi-1 provides endogenous protection of liver and kidney from ER stress and IR injury. Analysis of components of the bi-1-dependent pathway for protection from IR injury may therefore reveal new strategies for organ preservation.