Summary We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.

2 SUMMARYWe previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.

Master transcription factors interact with DNA to establish cell-type identity and to regulate gene expression in mammalian cells1,2. The genome-wide map of these transcription factor binding sites has been termed the cistrome3. Here we show that the androgen receptor (AR) cistrome undergoes extensive reprogramming during prostate epithelial transformation in man. Using human prostate tissue, we observed a core set of AR binding sites that are consistently reprogrammed in tumors. FOXA1 and HOXB13, co-localized with the reprogrammed AR sites in human tumor tissue. Introduction of FOXA1 and HOXB13 into an immortalized prostate cell line reprogrammed the AR cistrome to resemble that of a prostate tumor, functionally linking these specific factors to AR reprogramming. These findings offer mechanistic insights into a key set of events that drive normal prostate epithelium towards transformation and establish the centrality of epigenetic reprogramming in human prostate tumorigenesis.

Unlike most tumor suppressor genes, the most common genetic alterations in tumor protein p53 (TP53) are missense mutations. Mutant p53 protein is often abundantly expressed in cancers and specific allelic variants exhibit dominant-negative or gain-of-function activities in experimental models. To gain a systematic view of p53 function, we interrogated loss-of-function screens conducted in hundreds of human cancer cell lines and performed TP53 saturation mutagenesis screens in an isogenic pair of TP53 wild-type and null cell lines. We found that loss or dominant-negative inhibition of wild-type p53 function reliably enhanced cellular fitness. By integrating these data with the Catalog of Somatic Mutations in Cancer (COSMIC) mutational signatures database, we developed a statistical model that describes the TP53 mutational spectrum as a function of the baseline probability of acquiring each mutation and the fitness advantage conferred by attenuation of p53 activity. Collectively, these observations show that widely-acting and tissue-specific mutational processes combine with phenotypic selection to dictate the frequencies of recurrent TP53 mutations.

Summary Increased androgen receptor (AR) activity drives therapeutic resistance in advanced prostate cancer. The most common resistance mechanism is amplification of this locus presumably targeting the AR gene. Here, we identify and characterize a somatically acquired AR enhancer located 650 kilobases centromeric to the AR. Systematic perturbation of this enhancer using genome editing decreased proliferation by suppressing AR levels. Insertion of an additional copy of this region sufficed to increase proliferation under low androgen conditions and to decrease sensitivity to enzalutamide. Epigenetic data generated in localized prostate tumors and benign specimens support the notion that this region is a developmental enhancer. Collectively, these observations underscore the importance of epigenomic profiling in primary specimens and the value of deploying genome editing to functionally characterize noncoding elements. More broadly, this work identifies a therapeutic vulnerability for targeting the AR and emphasizes the importance of regulatory elements as highly recurrent oncogenic drivers.

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Initiation and completion of DNA replication defines the beginning and ending of S phase of the cell cycle. Successful progression through S phase requires that replication be properly regulated and monitored to ensure that the entire genome is duplicated exactly once, without errors, in a timely fashion. Given the immense size and complexity of eukaryotic genomes, this presents a significant challenge for the cell. As a result, DNA replication has evolved into a tightly regulated process involving the coordinated action of numerous factors that function in all phases of the cell cycle. We will review our current understanding of these processes from the formation of prereplicative complexes in preparation for S phase to the series of events that culminate in the loading of DNA polymerases during S phase. We will incorporate structural data from archaeal and bacterial replication proteins and discuss their implications for understanding the mechanism of action of their corresponding eukaryotic homologues. We will also describe the concept of replication licensing which protects against genomic instability by limiting initiation events to once per cell cycle. Lastly, we will review our knowledge of checkpoint pathways that maintain the integrity of stalled forks and relay defects in replication to the rest of the cell cycle.

Cy or RXL motifs have been previously shown to be cyclin binding motifs found in a wide range of cyclinCdk interacting proteins. We report the first kinetic analysis of the contribution of a Cy motif on a substrate to phosphorylation by cyclin-dependent kinases. For both cyclin A-Cdk2 and cyclin E-Cdk2 enzymes, the presence of a Cy motif decreased the K m(peptide) 75-120-fold while the k cat remained unchanged. The large effect of the Cy motif on the K m(peptide) suggests that the Cy motif and (S/T)PX(K/R) together constitute a bipartite substrate recognition sequence for cyclin-dependent kinases. Systematic changes in the length of the linker between the Cy motif and the phosphoacceptor serine suggest that both sites are engaged simultaneously to the cyclin and the Cdk, respectively, and eliminate a "bind and release" mechanism to increase the local concentration of the substrate. PS100, a peptide containing a Cy motif, acts as a competitive inhibitor of cyclin-Cdk complexes with a 15-fold lower K i for cyclin E-Cdk2 than for cyclin A-Cdk2. These results provide kinetic proof that a Cy motif located a minimal distance from the SPXK is essential for optimal phosphorylation by Cdks and suggest that small chemicals that mimic the Cy motif would be specific inhibitors of substrate recognition by cyclin-dependent kinases.Timely progression through the cell cycle depends upon the well orchestrated activation and deactivation of cyclin-dependent kinases. Each of these kinases is active for only a short period of the cell cycle during which time it phosphorylates a number of substrates required for entry into the next phase of the cell cycle. Cyclin A-Cdk2 1 and cyclin E-Cdk2 both play a major role in the G 1 /S transition of the cell cycle by the phosphorylation of various substrates including pRb, E2F, and CDC6 (1, 2). Despite their critical role in this process, little is known about how these substrates are targeted to specific cyclin-Cdk complexes.Because the (S/T)PX(K/R) consensus phosphorylation site is broadly applicable to all substrates of all Cdks, it would be incapable of conferring the substrate specificity seen within a cellular context. An alternate mechanism by which this specificity could be achieved is through the presence of a docking site on the substrate that recruits the appropriate cyclin-Cdk to the protein. The resulting high localized concentration of the cyclin-Cdk then facilitates the phosphorylation of potential SerThr phosphorylation sites that have been brought into close proximity. In previous work, we and others have identified a sequence motif present in a number of cellular proteins that interact with cyclin-Cdk complexes and could potentially perform this function (3-5). These cyclin binding motifs (Cy or RXL) have been found in substrates such as E2F and CDC6, activators like Cdc25a and inhibitors of the p21-27 family, and are absolutely required for the association of cyclin-Cdk complexes with these proteins (3, 4, 6 -8). The importance of this motif in the interaction of these prot...