Deregulated ribosomal RNA synthesis is associated with uncontrolled cancer cell proliferation. RNA polymerase (Pol) I, the multiprotein complex that synthesizes rRNA, is activated widely in cancer. Thus, selective inhibitors of Pol I may offer a general therapeutic strategy to block cancer cell proliferation. Coupling medicinal chemistry efforts to tandem cell-and molecular-based screening led to the design of CX-5461, a potent small-molecule inhibitor of rRNA synthesis in cancer cells. CX-5461 selectively inhibits Pol I-driven transcription relative to Pol II-driven transcription, DNA replication, and protein translation. Molecular studies demonstrate that CX-5461 inhibits the initiation stage of rRNA synthesis and induces both senescence and autophagy, but not apoptosis, through a p53-independent process in solid tumor cell lines. CX-5461 is orally bioavailable and demonstrates in vivo antitumor activity against human solid tumors in murine xenograft models. Our findings position CX-5461 for investigational clinical trials as a potent, selective, and orally administered agent for cancer treatment.

Hallmark deregulated signaling in cancer cells drives exces-

Abnormal accumulation of ␤-amyloid (A␤) in Alzheimer's disease (AD) is associated with prominent brain inflammation. Whereas earlier studies concluded that this inflammation is detrimental, more recent animal data suggest that at least some inflammatory processes may be beneficial and promote A␤ clearance. Consistent with these observations, overproduction of transforming growth factor (TGF)-␤1 resulted in a vigorous microglial activation that was accompanied by at least a 50% reduction in A␤ accumulation in human amyloid precursor protein (hAPP) transgenic mice. In a search for inflammatory mediators associated with this reduced pathology, we found that brain levels of C3, the central component of complement and a key inflammatory protein activated in AD, were markedly higher in hAPP͞TGF-␤1 mice than in hAPP mice. To assess the importance of complement in the pathogenesis of AD-like disease in mice, we inhibited C3 activation by expressing soluble complement receptor-related protein y (sCrry), a complement inhibitor, in the brains of hAPP mice. A␤ deposition was 2-to 3-fold higher in 1-year-old hAPP͞sCrry mice than in age-matched hAPP mice and was accompanied by a prominent accumulation of degenerating neurons. These results indicate that complement activation products can protect against A␤-induced neurotoxicity and may reduce the accumulation or promote the clearance of amyloid and degenerating neurons. These findings provide evidence for a role of complement and innate immune responses in AD-like disease in mice and support the concept that certain inflammatory defense mechanisms in the brain may be beneficial in neurodegenerative disease.

Alzheimer's disease (AD) is characterized by progressive neurodegeneration and cerebral accumulation of the β-amyloid peptide (Aβ), but it is unknown what makes neurons susceptible to degeneration. We report that the TGF-β type II receptor (TβRII) is mainly expressed by neurons, and that TβRII levels are reduced in human AD brain and correlate with pathological hallmarks of the disease. Reducing neuronal TGF-β signaling in mice resulted in age-dependent neurodegeneration and promoted Aβ accumulation and dendritic loss in a mouse model of AD. In cultured cells, reduced TGF-β signaling caused neuronal degeneration and resulted in increased levels of secreted Aβ and β-secretase-cleaved soluble amyloid precursor protein. These results show that reduced neuronal TGF-β signaling increases age-dependent neurodegeneration and AD-like disease in vivo. Increasing neuronal TGF-β signaling may thus reduce neurodegeneration and be beneficial in AD. IntroductionAlzheimer's disease (AD) is a progressive neurodegenerative disease that leads to loss of cognitive function in a large number of elderly people. The human AD brain is characterized by the accumulation of β-amyloid peptide (Aβ) in extracellular plaques and hyperphosphorylated tau in intracellular neurofibrillary tangles. In addition, there is degeneration of synapses and dendrites and a progressive loss of neurons involved in memory processes (1). The cause of this degeneration in AD remains unknown, and no effective treatments are available.Survival of neurons is dependent on extracellular signals from neurotrophic factors and related factors with trophic activity (reviewed in ref.2). Levels of the neurotrophin nerve growth factor (NGF) and its receptor, tropomyosin receptor kinase A (TRKA), as well as levels of brain-derived neurotrophic factor (BDNF) and its receptor, TRKB, are lower in human AD brains than in nondemented controls (2-5). It was therefore proposed that a deficiency in neurotrophic factor signaling would promote neurodegeneration and cognitive dysfunction in AD, but this hypothesis has not been tested using specific genetic inhibition of neurotrophic factor signaling in a mouse model for AD (reviewed in refs. 2, 6).

Patients with the most common and aggressive form of high-grade glioma, glioblastoma multiforme, have poor prognosis and few treatment options. In 2 immunocompetent mouse brain tumor models (CT26-BALB/c and Tu-2449-B6C3F1), we showed that a nonlytic retroviral replicating vector (Toca 511) stably delivers an optimized cytosine deaminase prodrug activating gene to the tumor lesion and leads to long-term survival after treatment with 5-fluorocytosine (5-FC). Survival benefit is dose dependent for both vector and 5-FC, and as few as 4 cycles of 5-FC dosing after Toca 511 therapy provides significant survival advantage. In the virally permissive CT26-BALB/c model, spread of Toca 511 to other tissues, particularly lymphoid tissues, is detectable by polymerase chain reaction (PCR) over a wide range of levels. In the Tu-2449-B6C3F1 model, Toca 511 PCR signal in nontumor tissues is much lower, spread is not always observed, and when observed, is mainly detected in lymphoid tissues at low levels. The difference in vector genome spread correlates with a more effective antiviral restriction element, APOBEC3, present in the B6C3F1 mice. Despite these differences, neither strain showed signs of treatment-related toxicity. These data support the concept that, in immunocompetent animals, a replicating retroviral vector carrying a prodrug activating gene (Toca 511) can spread through a tumor mass, leading to selective elimination of the tumor after prodrug administration, without local or systemic pathology. This concept is under investigation in an ongoing phase I/II clinical trial of Toca 511 in combination with 5-FC in patients with recurrent high-grade glioma ( NCT01156584).

Mitochondrial dysfunction is believed to participate in Huntington's disease (HD)

Retroviral replicating vectors (RRVs) are a nonlytic alternative to oncolytic replicating viruses as anticancer agents, being selective both for dividing cells and for cells that have defects in innate immunity and interferon responsiveness. Tumor cells fit both these descriptions. Previous publications have described a prototype based on an amphotropic murine leukemia virus (MLV), encoding yeast cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine (5-FC) to the potent anticancer drug, 5-fluorouracil (5-FU) in an infected tumor. We report here the selection of one lead clinical candidate based on a general design goal to optimize the genetic stability of the virus and the CD activity produced by the delivered transgene. Vectors were tested for titer, genetic stability, CD protein and enzyme activity, ability to confer susceptibility to 5-FC, and preliminary in vivo antitumor activity and stability. One vector, Toca 511, (aka T5.0002) encoding an optimized CD, shows a threefold increased specific activity in infected cells over infection with the prototype RRV and shows markedly higher genetic stability. Animal testing demonstrated that Toca 511 replicates stably in human tumor xenografts and, after 5-FC administration, causes complete regression of such xenografts. Toca 511 (vocimagene amiretrorepvec) has been taken forward to preclinical and clinical trials.

Spinocerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by specific degeneration of cerebellar, brainstem, and retinal neurons. Although they share little sequence homology, proteins implicated in polyQ disorders have common properties beyond their characteristic polyQ tract. These include the production of proteolytic fragments, nuclear accumulation, and processing by caspases. Here we report that ataxin-7 is cleaved by caspase-7, and we map two putative caspase-7 cleavage sites to Asp residues at positions 266 and 344 of the ataxin-7 protein. Site-directed mutagenesis of these two caspase-7 cleavage sites in the polyQ-expanded form of ataxin-7 produces an ataxin-7 D266N/D344N protein that is resistant to caspase cleavage. Although ataxin-7 displays toxicity, forms nuclear aggregates, and represses transcription in human embryonic kidney 293T cells in a polyQ length-dependent manner, expression of the non-cleavable D266N/D344N form of polyQ-expanded ataxin-7 attenuated cell death, aggregate formation, and transcriptional interference. Expression of the caspase-7 truncation product of ataxin-7-69Q or -92Q, which removes the putative nuclear export signal and nuclear localization signals of ataxin-7, showed increased cellular toxicity. We also detected N-terminal polyQ-expanded ataxin-7 cleavage products in SCA7 transgenic mice similar in size to those generated by caspase-7 cleavage. In a SCA7 transgenic mouse model, recruitment of caspase-7 into the nucleus by polyQ-expanded ataxin-7 correlated with its activation. Our results, thus, suggest that proteolytic processing of ataxin-7 by caspase-7 may contribute to SCA7 disease pathogenesis. Spinocerebellar ataxia type 7 (SCA7)4 is an autosomal dominant polyglutamine disorder clinically characterized by progressive ataxia and blindness. In the disease state, highly specific cell death ultimately occurs, most notably in the photoreceptor cells of the retina, Purkinje cells, dentate nuclei, and granule cells of the cerebellum, inferior olivary nuclei, and pontine neurons (1-3). SCA7 is a member of a family of "polyglutamine disorders." The retinal degeneration distinguishes SCA7 from the other polyglutamine diseases, although like many of the other polyQ disease proteins, ataxin-7 is widely expressed throughout the central nervous system. These autosomal dominant neurodegenerative diseases include Huntington disease (4), spinocerebellar ataxias type 1, 2, 3, 6, and 17 (SCA1, SCA2, SCA3 or Machado-Joseph disease, SCA6, SCA17) (5-8), dentatorubropallidoluysian atrophy (9), and spinal bulbar muscular atrophy (Kennedy disease) (10). The resultant increase in polyQ tract length derived from the CAG expansion within the gene product appears to exert direct toxicity on neuronal populations. Common mechanisms of disease pathogenesis include transcriptional dysregulation (11, 12) and proteolysis to produce toxic fragments (13-18). Neurodegeneration of specific neuronal populations found for each disease has been proposed to involve cell-specific...