Early patient stratification and predictive biomarkers in drug discovery and development: a case study of ulcerative colitis anti-TNF therapy.

The current drug discovery paradigm is long, costly, and prone to failure. For projects in early development, lack of efficacy in Phase II is a major contributor to the overall failure rate. Efficacy failures often occur from one of two major reasons: either the investigational agent did not achieve the required pharmacology or the mechanism targeted by the investigational agent did not significantly contribute to the disease in the tested patient population. The latter scenario can arise due to insufficient study power stemming from patient heterogeneity. If the subset of disease patients driven by the mechanism that is likely to respond to the drug can be identified and selected before enrollment begins, efficacy and response rates should improve. This will not only augment drug approval percentages, but will also minimize the number of patients at risk of side effects in the face of a suboptimal response to treatment. Here we describe a systems biology approach using molecular profiling data from patients at baseline for the development of predictive biomarker content to identify potential responders to a molecular targeted therapy before the drug is tested in humans. A case study is presented where a classifier to predict response to a TNF targeted therapy for ulcerative colitis is developed a priori and verified against a test set of patients where clinical outcomes are known. This approach will promote the tandem development of drugs with predictive response, patient selection biomarkers.

Construction of a computable cell proliferation network focused on non-diseased lung cells.

BACKGROUND: Critical to advancing the systems-level evaluation of complex biological processes is the development of comprehensive networks and computational methods to apply to the analysis of systems biology data (transcriptomics, proteomics/phosphoproteomics, metabolomics, etc.). Ideally, these networks will be specifically designed to capture the normal, non-diseased biology of the tissue or cell types under investigation, and can be used with experimentally generated systems biology data to assess the biological impact of perturbations like xenobiotics and other cellular stresses. Lung cell proliferation is a key biological process to capture in such a network model, given the pivotal role that proliferation plays in lung diseases including cancer, chronic obstructive pulmonary disease (COPD), and fibrosis. Unfortunately, no such network has been available prior to this work. RESULTS: To further a systems-level assessment of the biological impact of perturbations on non-diseased mammalian lung cells, we constructed a lung-focused network for cell proliferation. The network encompasses diverse biological areas that lead to the regulation of normal lung cell proliferation (Cell Cycle, Growth Factors, Cell Interaction, Intra- and Extracellular Signaling, and Epigenetics), and contains a total of 848 nodes (biological entities) and 1597 edges (relationships between biological entities). The network was verified using four published gene expression profiling data sets associated with measured cell proliferation endpoints in lung and lung-related cell types. Predicted changes in the activity of core machinery involved in cell cycle regulation (RB1, CDKN1A, and MYC/MYCN) are statistically supported across multiple data sets, underscoring the general applicability of this approach for a network-wide biological impact assessment using systems biology data. CONCLUSIONS: To the best of our knowledge, this lung-focused Cell Proliferation Network provides the most comprehensive connectivity map in existence of the molecular mechanisms regulating cell proliferation in the lung. The network is based on fully referenced causal relationships obtained from extensive evaluation of the literature. The computable structure of the network enables its application to the qualitative and quantitative evaluation of cell proliferation using systems biology data sets. The network is available for public use.

The perichromosomal layer.

In addition to genetic information, mitotic chromosomes transmit essential components for nuclear assembly and function in a new cell cycle. A specialized chromosome domain, called the perichromosomal layer, perichromosomal sheath, chromosomal coat, or chromosome surface domain, contains proteins required for a variety of cellular processes, including the synthesis of messenger RNA, assembly of ribosomes, repair of DNA double-strand breaks, telomere maintenance, and apoptosis regulation. The layer also contains many proteins of unknown function and is a major target in autoimmune disease. Perichromosomal proteins are found along the entire length of chromosomes, excluding centromeres, where sister chromatids are paired and spindle microtubules attach. Targeting of proteins to the perichromosomal layer occurs primarily during prophase, and they generally remain associated until telophase. During interphase, perichromosomal proteins localize to nucleoli, the nuclear envelope, nucleoplasm, heterochromatin, centromeres, telomeres, and/or the cytoplasm. It has been suggested that the perichromosomal layer may contribute to chromosome structure, as several of the associated proteins have functions in chromatin remodeling during interphase. We review the identified proteins associated with this chromosome domain and briefly discuss their known functions during interphase and mitosis.

Relevance of histone acetylation and replication timing for deposition of centromeric histone CENP-A.

A centromere-specific variant of histone H3, centromere protein A (CENP-A), is a critical determinant of centromeric chromatin, and its location on the chromosome may determine centromere identity. To search for factors that direct CENP-A deposition at a specific chromosomal locus, we took advantage of the observation that CENP-A, when expressed at elevated levels, can get incorporated at ectopic sites on the chromosome, in addition to the centromere. As core histone hypoacetylation and DNA replication timing have been implicated as epigenetic factors that may be important for centromere identity, we hypothesized that the sites of preferential CENP-A deposition will be distinguished by these parameters. We found that, on human dicentric chromosomes, ectopically expressed CENP-A preferentially incorporates at the active centromere only, despite the fact that the levels of histone acetylation and replication timing were indistinguishable at the two centromeres. In CHO cells, ectopically expressed CENP-A is preferentially targeted to some, but not all telomeric regions. Again, these regions could not be distinguished from other telomeres by their acetylation levels or replication timing. Thus histone acetylation and replication timing are not sufficient for specifying the sites of CENP-A deposition and likely for centromere identity.

Localization of human SMC1 protein at kinetochores.

Proper cohesion of sister chromatids is prerequisite for correct segregation of chromosomes during cell division. The cohesin multiprotein complex, conserved in eukaryotes, is required for sister chromatid cohesion. Human cohesion is composed of a stable heterodimer of the structural maintenance of chromosomes (SMC) family proteins, hSMC1 and hSMC3, and non-SMC components, hRAD21 and SA1 (or SA2). In yeast, cohesion associates with chromosomes from late G1 to metaphase and is required for the establishment and maintenance of both chromosome arm and centromeric cohesion. However, in human cells, the majority of cohesion dissociates from chromosomes before mitosis. Although it was recently shown that a small amount of hRAD21 localizes to the centromeres during metaphase, the presence of other cohesion components at the centromere has not been demonstrated in human cells. Here we report the mitosis-specific localization of hSMC1 to the kinetochores. hSMC1 is targeted to the kinetochore region during prophase concomitant with kinetochore assembly and remains through anaphase. Importantly, hSMC1 is targeted only to the active centromere on dicentric chromosomes. These results suggest that hSMC1 is an integral component of the functional kinetochore structure during mitosis.