Identification of alternative pathways of caspase activation is an important step to develop new antitumor treatments. We report here the result of a screening with a small chemical library, the Developmental Therapeutics Program-National Cancer Institute ''challenge set,'' on cells expressing mutated caspase-9. We have identified two molecules capable of activating an apoptosome-independent apoptotic pathway. These compounds, named F6 and G5, target the ubiquitinproteasome system by inhibiting the ubiquitin isopeptidases. We have shown that F6 and G5 induce a rather unique apoptotic pathway, which includes a Bcl-2-dependent but apoptosome-independent mitochondrial pathway with upregulation of the BH3-only protein Noxa, stabilization of the inhibitor of apoptosis antagonist Smac, but also the involvement of the death receptor pathway. Noxa plays an important role in the induction of mitochondrial fragmentation and caspase activation, whereas the death receptor pathway becomes critical in the absence of a functional apoptosome. This study suggests that screening of chemical libraries on cancer cells with defined mutations in apoptotic key elements can lead to the identification of compounds that are useful to characterize alternative pathways of caspase activation. (Cancer Res 2006; 66(18): 9235-44)

Missense point mutations in Gas3/PMP22 are responsible for the peripheral neuropathies Charcot-MarieTooth 1A and Dejerine Sottas syndrome. These mutations induce protein misfolding with the consequent accumulation of the proteins in the endoplasmic reticulum and the formation of aggresomes. During folding, Gas3/PMP22 associates with the lectin chaperone calnexin. Here, we show that calnexin interacts with the misfolded transmembrane domains of Gas3/PMP22, fused to green fluorescent protein, in a glycan-independent manner. In addition, photobleaching experiments in living cells revealed that Gas3/PMP22-green fluorescent protein mutants are mobile but diffuse at almost half the diffusion coefficient of wild type protein. Our results support emerging models for a glycan-independent chaperone role for calnexin and for the mechanism of retention of misfolded membrane proteins in the endoplasmic reticulum.

Several studies have indicated that proteasome inhibitors (PIs) are promising anticancer agents. We have discovered that PIs have the unique ability to activate effector caspases through a mitochondrial Bcl-2 inhibitable but caspase-9 independent pathway. Stabilization of released Smac induced by blockade of the proteasome could explain the apoptosome-independent cell death induced by PIs. Infact, Smac/ DIABLO critically supports this PIs-dependent caspase activation. By using a new assay, we confirm that at a single cell level both Smac and PIs can activate caspases in the absence of the apoptosome. Moreover, we have observed two PIs-induced kinetics of caspase activation, with caspase-9 being still required for the rapid caspase activation in response to mitochondrial depolarization, but dispensable for the slow DEVDase activation. In summary, our data indicate that PIs can activate downstream caspases at least in part through Smac/DIABLO stabilization.

Inhibitors of the ubiquitin-proteasome system (UPSIs) promote apoptosis of cancer cells and show encouraging anti-tumor activities in vivo. In this study, we evaluated the death activities of two different UPSIs: bortezomib and the isopeptidase inhibitor G5. To unveil whether these compounds elicit different types of death, we compared their effect both on apoptosis-proficient wild type mouse embryo fibroblasts and on cells defective for apoptosis (double-deficient Bax/Bak mouse embryo fibroblasts) (double knockout; DKO). We have discovered that (i) both inhibitors induce apoptosis in a Bax and Bak-dependent manner, (ii) both inhibitors elicit autophagy in WT and DKO cells, and (iii) only G5 can kill apoptosis-resistant DKO cells by activating a necrotic response. The induction of necrosis was confirmed by different experimental approaches, including time lapse analysis, HMGB1 release, and electron microscopy studies. Neither treatment with antinecrotic agents, such as antioxidants, poly(ADP-ribose) polymerase and JNK inhibitors, necrostatin, and the intracellular Ca 2؉ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester, nor overexpression of Bcl-2 and Bcl-xL prevented necrosis induced by G5. This necrotic death is characterized by the absence of protein oxidation and by the rapid cyclosporin A-independent dissipation of the mitochondrial membrane potential. Notably, a peculiar feature of the G5-induced necrosis is an early and dramatic reorganization of the actin cytoskeleton, coupled to an alteration of cell adhesion. The importance of cell adhesion impairment in the G5-induced necrotic death of DKO cells was confirmed by the antagonist effect of the extracellular matrixadhesive components, collagen and fibronectin.

From the nucleus, histone deacetylase 4 (HDAC4) regulates a variety of cellular processes, including growth, differentiation, and survival, by orchestrating transcriptional changes. Extracellular signals control its repressive influence mostly through regulating its nuclear-cytoplasmic shuttling. In particular, specific posttranslational modifications such as phosphorylation and caspase-mediated proteolytic processing operate on HDAC4 to promote its nuclear accumulation or export. To understand the signaling properties of this deacetylase, we investigated its cell death-promoting activity and the transcriptional repression potential of different mutants that accumulate in the nucleus. Here we show that, compared to that of other nuclear forms of HDAC4, a caspase-generated nuclear fragment exhibits a stronger cell death-promoting activity coupled with increased repressive effect on Runx2-or SRF-dependent transcription. However, this mutant displays reduced repressive action on MEF2C-driven transcription. Photobleaching experiments and quantitative analysis of the raw data, based on a two-binding-state compartmental model, demonstrate the existence of two nuclear pools of HDAC4 with different chromatin-binding properties. The caspase-generated fragment is weakly bound to chromatin, whereas an HDAC4 mutant defective in 14-3-3 binding or the wild-type HDAC5 protein forms a more stable complex. The tightly bound species show an impaired ability to induce cell death and repress Runx2-or SRF-dependent transcription less efficiently. We propose that, through specific posttranslation modifications, extracellular signals control two distinct nuclear pools of HDAC4 to differentially dictate cell death and differentiation. These two nuclear pools of HDAC4 are characterized by different repression potentials and divergent dynamics of chromatin interaction.