Systematic identification of genomic markers of drug sensitivity in cancer cells.

Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers for responses to targeted agents. Here, to uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines--which represent much of the tissue-type and genetic diversity of human cancers--with 130 drugs under clinical and preclinical investigation. In aggregate, we found that mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing's sarcoma cells harbouring the EWS (also known as EWSR1)-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.

Loss of function of ATXN1 increases amyloid beta-protein levels by potentiating beta-secretase processing of beta-amyloid precursor protein.

Alzheimer disease (AD) is a devastating neurodegenerative disease with complex and strong genetic inheritance. Four genes have been established to either cause familial early onset AD (APP, PSEN1, and PSEN2) or to increase susceptibility for late onset AD (APOE). To date approximately 80% of the late onset AD genetic variance remains elusive. Recently our genome-wide association screen identified four novel late onset AD candidate genes. Ataxin 1 (ATXN1) is one of these four AD candidate genes and has been indicated to be the disease gene for spinocerebellar ataxia type 1, which is also a neurodegenerative disease. Mounting evidence suggests that the excessive accumulation of Abeta, the proteolytic product of beta-amyloid precursor protein (APP), is the primary AD pathological event. In this study, we ask whether ATXN1 may lead to AD pathogenesis by affecting Abeta and APP processing utilizing RNA interference in a human neuronal cell model and mouse primary cortical neurons. We show that knock-down of ATXN1 significantly increases the levels of both Abeta40 and Abeta42. This effect could be rescued with concurrent overexpression of ATXN1. Moreover, overexpression of ATXN1 decreased Abeta levels. Regarding the underlying molecular mechanism, we show that the effect of ATXN1 expression on Abeta levels is modulated via beta-secretase cleavage of APP. Taken together, ATXN1 functions as a genetic risk modifier that contributes to AD pathogenesis through a loss-of-function mechanism by regulating beta-secretase cleavage of APP and Abeta levels.

Non-viral adeno-associated virus-based platform for stable expression of antibody combination therapeutics.

Antibody combination therapeutics (ACTs) are polyvalent biopharmaceuticals that are uniquely suited for the control of complex diseases, including antibiotic resistant infectious diseases, autoimmune disorders and cancers. However, ACTs also represent a distinct manufacturing challenge because the independent manufacture and subsequent mixing of monoclonal antibodies quickly becomes cost prohibitive as more complex mixtures are envisioned. We have developed a virus-free recombinant protein expression platform based on adeno-associated viral (AAV) elements that is capable of rapid and consistent production of complex antibody mixtures in a single batch format. Using both multiplexed immunoassays and cation exchange (CIEX) chromatography, cell culture supernatants generated using our system were assessed for stability of expression and ratios of the component antibodies over time. Cultures expressing combinations of three to ten antibodies maintained consistent expression levels and stable ratios of component antibodies for at least 60 days. Cultures showed remarkable reproducibility following cell banking, and AAV-based cultures showed higher stability and productivity than non-AAV based cultures. Therefore, this non-viral AAV-based expression platform represents a predictable, reproducible, quick and cost effective method to manufacture or quickly produce for preclinical testing recombinant antibody combination therapies and other recombinant protein mixtures.

Amyloid-ß production via cleavage of amyloid-ß protein precursor is modulated by cell density.

Mounting evidence suggests that Alzheimer's disease (AD) is caused by the accumulation of the small peptide, amyloid-ß (Aß), a proteolytic cleavage product of amyloid-ß protein precursor (AßPP). Aß is generated through a serial cleavage of AßPP by ß- and γ-secretase. Aß40 and Aß42 are the two main components of amyloid plaques in AD brains, with Aß42 being more prone to aggregation. AßPP can also be processed by α-secretase, which cleaves AßPP within the Aß sequence, thereby preventing the generation of Aß. Little is currently known regarding the effects of cell density on AßPP processing and Aß generation. Here we assessed the effects of cell density on AßPP processing in neuronal and non-neuronal cell lines, as well as mouse primary cortical neurons. We found that decreased cell density significantly increases levels of Aß40, Aß42, total Aß, and the ratio of Aß42: Aß40. These results also indicate that cell density is a significant modulator of AßPP processing. Overall, these findings carry profound implications for both previous and forthcoming studies aiming to assess the effects of various conditions and genetic/chemical factors, e.g., novel drugs on AßPP processing and Aß generation in cell-based systems. Moreover, it is interesting to speculate whether cell density changes in vivo may also affect AßPP processing and Aß levels in the AD brain.