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Aurora kinases play a key role in mitotic progression. Over-expression of Aurora kinases is found in several human cancers and correlated with histological malignancy and clinical outcomes. Therefore, Aurora kinase inhibitors should be useful in the treatment of cancers. Cell-based screening methods have an advantage over biochemical approaches because hits can be optimized to inhibit targets in the proper intracellular context. We developed a novel Aurora kinase inhibitor R763/AS703569 using an image-based phenotypic screen. The anti-proliferative effect was examined in a panel of tumor cell lines and primary cells. The efficacy was determined in a broad panel of xenograft models. R763/AS703569 inhibits Aurora kinases, along with a limited number of other kinases including FMS-related tyrosine kinase 3 (FLT3), and has potent anti-proliferative activity against many cell types accompanying unique phenotypic changes such as enlarged cell size, endoreduplication and apoptosis. The endoreduplication cycle induced by R763/AS703569 was irreversible even after the compound was withdrawn from the culture. Oral administration of R763/AS703569 demonstrated marked inhibition of tumor growth in xenograft models of pancreatic, breast, colon, ovarian, and lung tumors and leukemia. An acute myeloid leukemia cell line MV4-11, which carries a FLT3 internal tandem duplication mutation, is particularly sensitive to R763/AS703569 in vivo. R763/AS703569 is a potent inhibitor of Aurora kinases and exhibited significant anti-proliferative activity against a wide range of tumor cells both in vitro and in vivo. Inhibition of Aurora kinases has the potential to be a new addition to the treatment of cancers.

Nat. Genet. 46, 722–725 (2014); published online 25 May 2014; corrected after print 12 November 2014 In the version of this article initially published, the introductory paragraph mistakenly stated that ameloblasts were “cells in the tooth roots of the upper (maxilla) and lower (mandible) jaw responsible for depositing enamel during tooth development (odontogenesis).

Molecular analysis of archival tumor specimens has accelerated the discovery of clinically useful biomarkers. RNA-seq of RNA extracted from formalin fixed, paraffin-embedded (FFPE) specimens, a potentially valuable source of biomarkers, is hampered by the presence of highly abundant ribosomal RNA (rRNA), which is difficult to remove due to extensive RNA degradation. Here we report RNA-seq results from human FFPE samples. RNA was extracted from estrogen receptor positive (ER+) and negative (ER-) FFPE breast tumors 7-8 years after fixation and embedding. Sequencing libraries were prepared using a proprietary protocol with known strand direction. On average, 14 million reads were obtained from each lane of the Illumina GAII. After employing a proprietary rRNA depletion method, 18S and 28S rRNA are reduced to less than 5% of total mapped reads. Approximately 25 000 unique Ref-seq gene transcripts were detected in each library, demonstrating detection of most rare and intermediate transcripts as well as abundantly expressed transcripts. The relative abundance of the estrogen receptor gene ESR1 and co-expressed transcripts in the ESR1+ and ESR1- tumors correlated with expression differences measured by qPCR. Directional analysis indicated that 95% of the reads mapped to known genes had the correct direction, whereas remaining 5% with antisense direction, which possibly serve as regulatory functions. Among the uniquely mapped reads, ~ 25% were within known exons, 41% in introns, and 34% in intergenic regions. 'Valleys' and 'hills' were commonly observed across the three mapped regions. A proprietary region analysis method was developed to identify any region (not limited to known Ref-seqs) exhibiting differential expression. Using this method, we identified 34 regions that were highly differentially expressed between the ESR1+ and ESR1- tumor specimens. These regions represent exons, introns and intergenic areas. Our findings were confirmed by qPCR. These preliminary studies demonstrate the feasibility of using RNA-seq with FFPE tumor specimens. We anticipate that RNA-seq will provide a new perspective about transcriptional changes that underlie the phenotypes of individual tumors.

The activation of T cells, mediated by the T-cell receptor (TCR), activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation. In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 × 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLCγ1 and SHP-1 (PTP1C) as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1), transmembrane molecules (EDG1, IL-10Rα and integrin α2), cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1), and cytoskeletal molecules (moesin and vimentin). Furthermore, using truncated Lck, PLCγ1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes. This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings.