Haley Hieronymus

The Connectivity Map: Using Gene-Expression Signatures to Connect Small Molecules, Genes, and Disease

To pursue a systematic approach to the discovery of functional connections among diseases, genetic perturbation, and drug action, we have created the first installment of a reference collection of gene-expression profiles from cultured human cells treated with bioactive small molecules, together with pattern-matching software to mine these data. We demonstrate that this “Connectivity Map” resource can be used to find connections among small molecules sharing a mechanism of action, chemicals and physiological processes, and diseases and drugs. These results indicate the feasibility of the approach and suggest the value of a large-scale community Connectivity Map project.

Integrative Genomic Profiling of Human Prostate Cancer

Annotation of prostate cancer genomes provides a foundation for discoveries that can impact disease understanding and treatment. Concordant assessment of DNA copy number, mRNA expression, and focused exon resequencing in 218 prostate cancer tumors identified the nuclear receptor coactivator NCOA2 as an oncogene in approximately 11% of tumors. Additionally, the androgen-driven TMPRSS2-ERG fusion was associated with a previously unrecognized, prostate-specific deletion at chromosome 3p14 that implicates FOXP1, RYBP, and SHQ1 as potential cooperative tumor suppressors. DNA copy-number data from primary tumors revealed that copy-number alterations robustly define clusters of low- and high-risk disease beyond that achieved by Gleason score. The genomic and clinical outcome data from these patients are now made available as a public resource.

COT drives resistance to RAF inhibition through MAP kinase pathway reactivation

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50–70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma—an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative ‘druggable’ targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.

Reciprocal Feedback Regulation of PI3K and Androgen Receptor Signaling in PTEN-Deficient Prostate Cancer

Prostate cancer is characterized by its dependence on androgen receptor (AR) and frequent activation of PI3K signaling. We find that AR transcriptional output is decreased in human and murine tumors with PTEN deletion and that PI3K pathway inhibition activates AR signaling by relieving feedback inhibition of HER kinases. Similarly, AR inhibition activates AKT signaling by reducing levels of the AKT phosphatase PHLPP. Thus, these two oncogenic pathways cross-regulate each other by reciprocal feedback. Inhibition of one activates the other, thereby maintaining tumor cell survival. However, combined pharmacologic inhibition of PI3K and AR signaling caused near-complete prostate cancer regressions in a Pten-deficient murine prostate cancer model and in human prostate cancer xenografts, indicating that both pathways coordinately support survival.

Genome-wide localization of the nuclear transport machinery couples transcriptional status and nuclear organization

The association of genes with the nuclear pore complex (NPC) and nuclear transport factors has been implicated in transcriptional regulation. We therefore examined the association of components of the nuclear transport machinery including karyopherins, nucleoporins, and the Ran guanine-nucleotide exchange factor (RanGEF) with the Saccharomyces cerevisiae genome. We find that most nucleoporins and karyopherins preferentially associate with a subset of highly transcribed genes and with genes that possess Rap1 binding sites whereas the RanGEF preferentially associates with transcriptionally inactive genes. Consistent with coupling of transcription to the nuclear pore, we show that transcriptional activation of the GAL genes results in their association with nuclear pore proteins, relocation to the nuclear periphery, and loss of RanGEF association. Taken together, these results indicate that the organization of the genome is coupled via transcriptional state to the nuclear transport machinery.

Cooperativity of TMPRSS2-ERG with PI3-kinase pathway activation in prostate oncogenesis

The TMPRSS2-ERG fusion, present in approximately 50% of prostate cancers, is less common in prostatic intraepithelial neoplasia (PIN), raising questions about whether TMPRSS2-ERG contributes to disease initiation. We identified the translational start site of a common TMPRSS2-ERG fusion and showed that transgenic TMPRSS2-ERG mice develop PIN, but only in the context of PI3-kinase pathway activation. TMPRSS2-ERG–positive human tumors are also enriched for PTEN loss, suggesting cooperation in prostate tumorigenesis.

FAS and NF-κB signalling modulate dependence of lung cancers on mutant EGFR

Human lung adenocarcinomas with activating mutations in EGFR (epidermal growth factor receptor) often respond to treatment with EGFR tyrosine kinase inhibitors (TKIs), but the magnitude of tumour regression is variable and transient. This heterogeneity in treatment response could result from genetic modifiers that regulate the degree to which tumour cells are dependent on mutant EGFR. Through a pooled RNA interference screen, we show that knockdown of FAS and several components of the NF-κB pathway specifically enhanced cell death induced by the EGFR TKI erlotinib in EGFR-mutant lung cancer cells. Activation of NF-κB through overexpression of c-FLIP or IKK (also known as CFLAR and IKBKB, respectively), or silencing of IκB (also known as NFKBIA), rescued EGFR-mutant lung cancer cells from EGFR TKI treatment. Genetic or pharmacologic inhibition of NF-κB enhanced erlotinib-induced apoptosis in erlotinib-sensitive and erlotinib-resistant EGFR-mutant lung cancer models. Increased expression of the NF-κB inhibitor IκB predicted for improved response and survival in EGFR-mutant lung cancer patients treated with EGFR TKI. These data identify NF-κB as a potential companion drug target, together with EGFR, in EGFR-mutant lung cancers and provide insight into the mechanisms by which tumour cells escape from oncogene dependence.

A public genome-scale lentiviral expression library of human ORFs

Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.

Genome-wide analysis of RNA–protein interactions illustrates specificity of the mRNA export machinery

Nuclear export of mRNA is mediated by a complex machinery of RNA-binding proteins that recognizes and routes mRNAs through a messenger ribonucleoprotein (mRNP) network. The full spectrum of mRNA cargoes for any dedicated mRNA export factor is unknown. We identified the mRNAs that bind two conserved yeast mRNA export factors, Yra1 (refs. 1–5) and Mex67 (refs. 6,7), on a genome-wide scale and determined their level of binding. Yra1 and Mex67 bind approximately 1,000 and 1,150 mRNAs, respectively, corresponding to almost 20% of the yeast genome and roughly 36% of all transcriptional events each. The binding level of Yra1 targets is related to their transcriptional frequency, but that of Mex67 targets is not. Yra1-bound transcripts are enriched in mRNAs that are regulated by a number of transcription factors. Yra1- and Mex67-bound populations also show enrichment of mRNAs encoding distinct functional classes of proteins, some of which are regulated by these transcription factors. We determined that one such transcription factor, Abf1 (refs. 8–10), associates with Yra1. These results indicate a previously unidentified specificity of mRNA export factors, which coordinates the export of transcriptionally co-regulated, functional classes of transcripts, perhaps through interactions with the transcriptional machinery.

Bead-based profiling of tyrosine kinase phosphorylation identifies SRC as a potential target for glioblastoma therapy

The aberrant activation of tyrosine kinases represents an important oncogenic mechanism, and yet the majority of such events remain undiscovered. Here we describe a bead-based method for detecting phosphorylation of both wild-type and mutant tyrosine kinases in a multiplexed, high-throughput and low-cost manner. With the aim of establishing a tyrosine kinase–activation catalog, we used this method to profile 130 human cancer lines. Follow-up experiments on the finding that SRC is frequently phosphorylated in glioblastoma cell lines showed that SRC is also activated in primary glioblastoma patient samples and that the SRC inhibitor dasatinib (Sprycel) inhibits viability and cell migration in vitro and tumor growth in vivo. Testing of dasatinib-resistant tyrosine kinase alleles confirmed that SRC is indeed the relevant target of dasatinib, which inhibits many tyrosine kinases. These studies establish the feasibility of tyrosine kinome–wide phosphorylation profiling and point to SRC as a possible therapeutic target in glioblastoma.