The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.translational research | PCI-32765 | Imbruvica

We predicted gene function using synthetic lethal genetic interactions between null alleles in Saccharomyces cerevisiae. Phenotypic and protein interaction data indicate that synthetic lethal gene pairs function in parallel or compensating pathways. Congruent gene pairs, defined as sharing synthetic lethal partners, are in single pathway branches. We predicted benomyl sensitivity and nuclear migration defects using congruence; these phenotypes were uncorrelated with direct synthetic lethality. We also predicted YLL049W as a new member of the dynein-dynactin pathway and provided new supporting experimental evidence. We performed synthetic lethal screens of the parallel mitotic exit network (MEN) and Cdc14 early anaphase release pathways required for late cell cycle. Synthetic lethal interactions bridged genes in these pathways, and high congruence linked genes within each pathway. Synthetic lethal interactions between MEN and all components of the Sin3/Rpd3 histone deacetylase revealed a novel function for Sin3/Rpd3 in promoting mitotic exit in parallel to MEN. These in silico methods can predict phenotypes and gene functions and are applicable to genomic synthetic lethality screens in yeast and analogous RNA interference screens in metazoans.

Cohesion establishment and maintenance are carried out by proteins that modify the activity of Cohesin, an essential complex that holds sister chromatids together. Constituents of the replication fork, such as the DNA polymerase ␣-binding protein Ctf4, contribute to cohesion in ways that are poorly understood. To identify additional cohesion components, we analyzed a ctf4⌬ synthetic lethal screen performed on microarrays. We focused on a subset of ctf4⌬-interacting genes with genetic instability of their own. Our analyses revealed that 17 previously studied genes are also necessary for the maintenance of robust association of sisters in metaphase. Among these were subunits of the MRX complex, which forms a molecular structure similar to Cohesin. Further investigation indicated that the MRX complex did not contribute to metaphase cohesion independent of Cohesin, although an additional role may be contributed by XRS2. In general, results from the screen indicated a sister chromatid cohesion role for a specific subset of genes that function in DNA replication and repair. This subset is particularly enriched for genes that support the S-phase checkpoint. We suggest that these genes promote and protect a chromatin environment conducive to robust cohesion. INTRODUCTIONIn budding yeast, Cohesin is a four subunit protein complex (Mcd1/Scc1, Scc3, Smc1, and Smc3) that depends on the activity of regulatory proteins for its chromosome association, activation, and destruction in each cell cycle (reviewed in Nasmyth, 2001). Sister chromatid association by Cohesin must be established during S phase (Skibbens et al., 1999;Toth et al., 1999) and is maintained until separation of sister chromatids at anaphase. Many studies indicate a role for the replication fork in establishment of robust sister chromatid cohesion, in addition to its traditional role of semiconservative DNA duplication. Several replication-associated proteins from budding yeast are known to support robust cohesion, including the products of nonessential genes CTF4, CTF8, CTF18, DCC1, TRF4, and TRF5 (Wang et al., 2000b;Hanna et al., 2001;Mayer et al., 2001). Null mutants for these genes lead to metaphase cohesion failure at frequencies of ϳ25-35% in cells that are held at metaphase in the absence of microtubules.These nonessential replication fork constituents represent several subcomplexes at the replication fork and contribute independent primary molecular functions that are not well understood. Ctf4 protein forms an association with DNA polymerase ␣ (Formosa and Nittis, 1999) that may compete with binding of a chromatin remodeling subunit Cdc68/ Pob3 (Wittmeyer and Formosa, 1997). Ctf18 protein is a component of an alternative RF-C complex in which it replaces Rfc1, and is joined by Ctf8 and Dcc1 subunits (Hanna et al., 2001;Mayer et al., 2001). The orthologous human RF-C CTF18 complex can load PCNA onto DNA and promote Pol␦ activity in vitro (Bermudez et al., 2003;Kanellis et al., 2003;Merkle et al., 2003). The nonessential proteins Trf4 and Trf5 together co...