BACKGROUND Cellular therapies could play a role in cancer treatment and regenerative medicine if it were possible to quickly eliminate the infused cells in case of adverse events. We devised an inducible T-cell safety switch that is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization. When exposed to a synthetic dimerizing drug, the inducible caspase 9 (iCasp9) becomes activated and leads to the rapid death of cells expressing this construct. METHODS We tested the activity of our safety switch by introducing the gene into donor T cells given to enhance immune reconstitution in recipients of haploidentical stem-cell transplants. Patients received AP1903, an otherwise bioinert small-molecule dimerizing drug, if graft-versus-host disease (GVHD) developed. We measured the effects of AP1903 on GVHD and on the function and persistence of the cells containing the iCasp9 safety switch. RESULTS Five patients between the ages of 3 and 17 years who had undergone stem-cell transplantation for relapsed acute leukemia were treated with the genetically modified T cells. The cells were detected in peripheral blood from all five patients and increased in number over time, despite their constitutive transgene expression. A single dose of dimerizing drug, given to four patients in whom GVHD developed, eliminated more than 90% of the modified T cells within 30 minutes after administration and ended the GVHD without recurrence. CONCLUSIONS The iCasp9 cell-suicide system may increase the safety of cellular therapies and expand their clinical applications. (Funded by the National Heart, Lung, and Blood Institute and the National Cancer Institute; ClinicalTrials.gov number, NCT00710892.)

Dimerization and oligomerization are general biological control mechanisms contributing to the activation of cell membrane receptors, transcription factors, vesicle fusion proteins, and other classes of intra- and extracellular proteins. Cell permeable, synthetic ligands were devised that can be used to control the intracellular oligomerization of specific proteins. To demonstrate their utility, these ligands were used to induce intracellular oligomerization of cell surface receptors that lacked their transmembrane and extracellular regions but contained intracellular signaling domains. Addition of these ligands to cells in culture resulted in signal transmission and specific target gene activation. Monomeric forms of the ligands blocked the pathway. This method of ligand-regulated activation and termination of signaling pathways has the potential to be applied wherever precise control of a signal transduction pathway is desired.

The efficacy of adoptive T-cell therapy as treatment for malignancies may be enhanced by genetic modification of infused cells. However, oncogenic events due to vector/transgene integration, and toxicities due to the infused cells themselves, have tempered enthusiasm. A safe and efficient means of removing aberrant cells in vivo would ameliorate these concerns. We describe a "safety switch" that can be stably and efficiently expressed in human T cells without impairing phenotype, function, or antigen specificity. This reagent is based on a modified human caspase 9 fused to a human FK506 binding protein (FKBP) to allow conditional dimerization using a small molecule pharmaceutical. A single 10-nM dose of synthetic dimerizer drug induces apoptosis in 99% of transduced cells selected for high transgene expression in vitro and in vivo. This system has several advantages over currently available suicide genes.

Fibroblast Growth Factor Receptor-1 (FGFR1) is commonly overexpressed in advanced prostate cancer (PCa). To investigate causality, we utilized an inducible FGFR1 (iFGFR1) prostate mouse model. Activation of iFGFR1 with chemical inducers of dimerization (CID) led to highly synchronous, step-wise progression to adenocarcinoma that is linked to an epithelial-to-mesenchymal transition (EMT). iFGFR1 inactivation by CID withdrawal led to full reversion of prostatic intraepithelial neoplasia, whereas PCa lesions became iFGFR1-independent. Gene expression profiling at distinct stages of tumor progression revealed an increase in EMT-associated Sox9 and changes in the Wnt signaling pathway, including Fzd4, which was validated in human PCa. The iFGFR1 model clearly implicates FGFR1 in PCa progression and demonstrates how CID-inducible models can help evaluate candidate molecules in tumor progression and maintenance.

A role for caspase-10, previously implicated in the autoimmune lymphoproliferative syndrome, in death receptor signaling has not been directly shown. Here we show that caspase-10 can function independently of caspase-8 in initiating Fas-and tumor necrosis factor-related apoptosis-inducing ligand-receptor-mediated apoptosis. Moreover, Fas crosslinking in primary human T cells leads to the recruitment and activation of caspase-10. Fluorescent resonance energy transfer analysis indicates that the death-effector domains of caspase-8 and -10 both interact with the death-effector domain of FADD. Nonetheless, we find that caspase-8 and -10 may have different apoptosis substrates and therefore potentially distinct roles in death receptor signaling or other cellular processes.D eath receptors (DRs) of the tumor necrosis factor receptor superfamily contain a conserved ''death domain'' in their intracellular region (1). Fas associates with an adaptor molecule, FADD, through homotypic death domain interactions (2, 3). FADD then recruits caspase-8 through homotypic interactions of death-effector domains (DEDs), leading to caspase-8 activation and apoptosis (4, 5). Other DRs may also signal by recruiting caspase-8 through FADD. Among at least 14 mammalian caspases identified so far, only caspase-10 shares homologous DEDs with caspase-8 (6, 7), suggesting that caspase-10 may also function by interacting with DRs. However, recent studies have failed to detect caspase-10 in DR signaling complexes (8, 9).We have studied patients with autoimmune lymphoproliferative syndrome, a genetic disorder of lymphocyte homeostasis and immune tolerance. Some of these patients harbor a dominant interfering mutation in caspase-10 (10). Caspase-10 abnormalities are associated with defective DR-mediated apoptosis in T cells and dendritic cells. Another homozygous-altered allele was found to be associated with autoimmune lymphoproliferative syndrome (10). Recently, this same mutant allele (V367I in caspase-10 S or V410I in caspase-10 L ) has been found to be present at high frequency in heterozygous form in certain populations (11). Given that this allele encodes a protein with reduced caspase activity, the wide occurrence in this allele may suggest a broader role as a susceptibility factor in immune diseases. Here we show that caspase-10 enters DR signaling complexes in primary T cells and that caspase-10 can functionally substitute for caspase-8, providing direct evidence for a physiological role of caspase-10 in DR signal transduction. Experimental ProceduresCell Line, Abs, and Plasmids. Wild-type Jurkat cells and caspase-8-deficient Jurkat mutant, I9.2, were kindly provided by John Blenis (12). Monoclonal anti-caspase-10 Ab specific for caspase-10 DEDs was from the Medical and Biological Laboratories (Nagoya, Japan). MAbs to caspase-3, caspase-8, RIP, and FADD were from PharMingen. Anti-APO1.3 (anti-Fas) was obtained from Kamiya Biomedical (Thousand Oaks, CA). Polyclonal rabbit anti-Fas Ab was from Marcus Peter (University of Chicago). Full-len...

Eukaryotic transcriptional activators mediate transcriptional induction through stabilization of the preinitiation complex, probably through direct interactions with basal transcription factors. In vitro studies on the role of an activator in the maintenance of on-going transcription (reinitiation) have been contradictory, suggesting that, after formation of a preinitiation complex, an activator may or may not be necessary for transcription to be maintained. We have developed a means of regulating transcription in living cells through the use of both homodimeric and heterodimerizing synthetic ligands that allow the ligand-dependent association and disassociation of a transcriptional activation domain with a promoter. Here we report that maintaining the transcription of endogenous genes in vivo, in both yeast and human cells, requires the continuous presence of the activation domain. The use of synthetic ligands as a transcriptional on-off switch represents a powerful means of controlling the transcription in vitro and in vivo for both experimental and therapeutic purposes.

The casp9 protein plays a critical role in apoptosis induced by a variety of death stimuli. A regulator of apoptosis, Apaf-1, binds to and activates pro-casp9 in the presence of cytochrome c and dATP, a requirement that is bypassed by deletion of the WD-40 repeats located in the C-terminal half of Apaf-1. In this report, we used constitutively active Apaf-1 mutant lacking the WD-40 repeat region to study the mechanism and regulation of pro-casp9 activation. Mutational analysis revealed that only a small portion of the CED-4 homologous region (residues 456 -559) could be deleted without destroying the ability of Apaf-1-(1-559) to activate pro-casp9. Apaf-1 can self-associate to form oligomers. Disruption of Apaf-1 self-association by deletion (⌬109 -559) or mutation of the P-loop region (K149R) abrogated Apaf-1-mediated pro-casp9 activation. Forced oligomerization of the caspase recruitment domain of Apaf-1 was sufficient for pro-casp9 activation. Dimerization of chimeric Fpkpro-casp9 protein with the dimerizer drug FK1012 induced pro-casp9 processing and apoptosis in cells. Significantly, the C-terminal region containing WD-40 repeats interacted with its N-terminal CED-4 homologous region, as determined by immunoprecipitation experiments. Importantly, expression of the WD-40 repeat region inhibited Apaf-1 self-association and proteolytic activation of pro-casp9. These studies provide a mechanism by which Apaf-1 promotes autoactivation of procasp9 through Apaf-1 self-association, a process that is negatively regulated by the WD-40 repeats.Programmed cell death, or apoptosis, an evolutionarily conserved and genetically regulated biological process, plays a critical role in development and tissue homeostasis of multicellular organisms (1-4). Dysfunction of this process contributes to the pathogenesis of several diseases in humans and mice (4 -6). A family of cysteine proteases (designated caspases) related to Caenorhabditis elegans CED-3 appears to represent the executionary arm of the apoptotic program. Each caspase contains conserved residues important for specific proteolytic activity cleaving substrates after aspartate residues (7-9). Caspases are synthesized in cells as inactive zymogens and, upon stimulation with apoptotic signals, are processed into mature forms composed of a tetramer of two large and two small subunits (10, 11). The apoptotic process is characterized by sequential activation of multiple caspases. Depending on their order of activation in the proteolytic cascade, caspases can be divided into upstream and downstream caspases. The casp9 protein is an upstream caspase that contains a caspase recruit domain (CARD) in its N terminus, a region that is also present in the prodomain of several death proteases including CED-3, casp1, and casp2 (12). Activation of casp9 initiates a protease cascade with subsequent activation of downstream casp3 and casp7, leading to cleavage of target proteins and execution of the apoptotic program (13).Our understanding of how caspases are activated in mammalian cell...

TMPRSS2/ERG gene fusions are found in the majority of prostate cancers; however, there is significant heterogeneity in the 5 ¶ region of the alternatively spliced fusion gene transcripts. We have found that there is also significant heterogeneity within the coding exons as well. There is variable inclusion of a 72-bp exon and other novel alternatively spliced isoforms. To assess the biological significance of these alternatively spliced transcripts, we expressed various transcripts in primary prostatic epithelial cells (PrEC) and in an immortalized PrEC line, PNT1a. The fusion gene transcripts promoted proliferation, invasion, and motility with variable activities that depended on the structure of the 5 ¶ region encoding the TMPRSS2/ERG fusion and the presence of the 72-bp exon. Cotransfection of different isoforms further enhanced biological activity, mimicking the situation in vivo, in which multiple isoforms are expressed. Finally, knockdown of the fusion gene in VCaP cells resulted in inhibition of proliferation in vitro and tumor progression in an in vivo orthotopic mice model. Our results indicate that TMPRSS2/ ERG fusion isoforms have variable biological activities promoting tumor initiation and progression and are consistent with our previous clinical observations indicating that certain TMPRSS2/ERG fusion isoforms are significantly correlated with more aggressive disease. [Cancer Res 2008; 68(20):8516-24]