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In prostate cancer, the development of castration resistance is pivotal in progression to aggressive disease. However, understanding of the pathways involved remains incomplete. In this study, we performed a high-throughput genetic screen to identify kinases that enable tumor formation by androgen-dependent prostate epithelial (LHSR-AR) cells under androgen-deprived conditions. In addition to the identification of known mediators of castration resistance, which served to validate the screen, we identified a mitotic-related serine/threonine kinase, NEK6, as a mediator of androgen-independent tumor growth. NEK6 was overexpressed in a subset of human prostate cancers. Silencing NEK6 in castration-resistant cancer cells was sufficient to restore sensitivity to castration in a mouse xenograft model system. Tumors in which castration resistance was conferred by NEK6 were predominantly squamous in histology with no evidence of AR signaling. Gene expression profiling suggested that NEK6 overexpression stimulated cytoskeletal, differentiation, and immune signaling pathways and maintained gene expression patterns normally decreased by castration. Phosphoproteome profiling revealed the transcription factor FOXJ2 as a novel NEK6 substrate, with FOXJ2 phosphorylation associated with increased expression of newly identified NEK6 transcriptional targets. Overall, our studies establish NEK6 signaling as a central mechanism mediating castration-resistant prostate cancer. Cancer Res; 77(3); 753–65. ©2016 AACR.

Description of additional methods and procedures used in the study.

GO terms for NEK6 upregulated genes Day 5 after castration (filtered for >10 GeneHits).

Legend for Supplemental Figures S1-S6.

To identify novel therapeutic targets in acute myeloid leukemia (AML), we examined kinase expression patterns in primary AML samples. We found that the serine/threonine kinase IKBKE, a noncanonical IkB kinase, is expressed at higher levels in myeloid leukemia cells compared with normal hematopoietic cells. Inhibiting IKBKE, or its close homolog TANK-binding kinase 1 (TBK1), by either short hairpin RNA knockdown or pharmacological compounds, induces apoptosis and reduces the viability of AML cells. Using gene expression profiling and gene set enrichment analysis, we found that IKBKE/TBK1-sensitive AML cells typically possess an MYC oncogenic signature. Consistent with this finding, the MYC oncoprotein was significantly downregulated upon IKBKE/TBK1 inhibition. Using proteomic analysis, we found that the oncogenic gene regulator YB-1 was activated by IKBKE/TBK1 through phosphorylation, and that YB-1 binds to the MYC promoter to enhance MYC gene transcription. Momelotinib (CYT387), a pharmacological inhibitor of IKBKE/TBK1, inhibits MYC expression, reduces viability and clonogenicity of primary AML cells, and demonstrates efficacy in a murine model of AML. Together, these data identify IKBKE/TBK1 as a promising therapeutic target in AML.

Alternative splicing of mRNA precursors represents a key gene expression regulatory step and permits the generation of distinct protein products with diverse functions. In a genome-scale expression screen for inducers of the epithelial-to-mesenchymal transition (EMT), we found a striking enrichment of RNA-binding proteins. We validated that QKI and RBFOX1 were necessary and sufficient to induce an intermediate mesenchymal cell state and increased tumorigenicity. Using RNA-seq and eCLIP analysis, we found that QKI and RBFOX1 coordinately regulated the splicing and function of the actin-binding protein FLNB, which plays a causal role in the regulation of EMT. Specifically, the skipping of FLNB exon 30 induced EMT by releasing the FOXC1 transcription factor. Moreover, skipping of FLNB exon 30 is strongly associated with EMT gene signatures in basal-like breast cancer patient samples. These observations identify a specific dysregulation of splicing, which regulates tumor cell plasticity and is frequently observed in human cancer., eLife digest As the human body develops, countless cells change from one state into another. Two important cell states are known as epithelial and mesenchymal. Cells in the epithelial state tend to be tightly connected and form barriers, like skin cells. Mesenchymal state cells are loosely organized, move around more and make up connective tissues. Some cells alternate between these states via an epithelial-to-mesenchymal transition (EMT for short) and back again. Without this transition, certain organs would not develop and wounds would not heal. Yet, cancer cells also use this transition to spread to distant sites of the body. Such cancers are often the most aggressive, and therefore the most deadly. The epithelial-to-mesenchymal transition is dynamically regulated in a reversible manner. For example, the genes for some proteins might only be active in the epithelial state and further reinforce this state by turning on other ‘epithelial genes’. Alternatively, there might be differences in the processing of mRNA molecules – the intermediate molecules between DNA and protein – that result in the production of different proteins in epithelial and mesenchymal cells. Li, Choi et al. wanted to know which of the thousands of human genes can endow epithelial state cells with mesenchymal characteristics. A better understanding of the switch could help to prevent cancers undergoing an epithelial-to-mesenchymal transition. From a large-scale experiment in human breast cancer cells, Li, Choi et al. found that a group of proteins that bind and modify mRNA molecules are important for the epithelial-to-mesenchymal transition. Two proteins in particular promoted the transition, most likely by binding to the mRNA of a third protein called FLNB and removing a small piece of it. FLNB normally works to prevent the epithelial-to-mesenchymal transition, but the smaller protein encoded by the shorter mRNA promoted the transition by turning on ‘mesenchymal genes’. This switching between different FLNB proteins happens in some of the more aggressive breast cancers, which also contain mesenchymal cells. Finding out which FLNB protein is made in a given cancer may provide an indication of its aggressiveness. Also, looking for drugs that can target the mRNA-binding proteins or FLNB may one day lead to new treatments for some of the most aggressive breast cancers.

The Janus kinases (JAKs) and their downstream effectors, signal transducer and activator of transcription proteins (STATs), form a critical immune cell signaling circuit, which is of fundamental importance in innate immunity, inflammation, and hematopoiesis, and dysregulation is frequently observed in immune disease and cancer. The high degree of structural conservation of the JAK ATP binding pockets has posed a considerable challenge to medicinal chemists seeking to develop highly selective inhibitors as pharmacological probes and as clinical drugs. Here we report the discovery and optimization of 2,4-substituted pyrimidines as covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Investigation of structure-activity relationship (SAR) utilizing biochemical and transformed Ba/F3 cellular assays resulted in identification of potent and selective inhibitors such as compounds 9 and 45. A 2.9 Å cocrystal structure of JAK3 in complex with 9 confirms the covalent interaction. Compound 9 exhibited decent pharmacokinetic properties and is suitable for use in vivo. These inhibitors provide a set of useful tools to pharmacologically interrogate JAK3-dependent biology.

In prostate cancer, the development of castration resistance is pivotal in progression to aggressive disease. However, understanding of the pathways involved remains incomplete. In this study, we performed a high-throughput genetic screen to identify kinases that enable tumor formation by androgen-dependent prostate epithelial (LHSR-AR) cells under androgen-deprived conditions. In addition to the identification of known mediators of castration resistance, which served to validate the screen, we identified a mitotic-related serine/threonine kinase, NEK6, as a mediator of androgen-independent tumor growth. NEK6 was overexpressed in a subset of human prostate cancers. Silencing NEK6 in castration-resistant cancer cells was sufficient to restore sensitivity to castration in a mouse xenograft model system. Tumors in which castration resistance was conferred by NEK6 were predominantly squamous in histology with no evidence of AR signaling. Gene expression profiling suggested that NEK6 overexpression stimulated cytoskeletal, differentiation, and immune signaling pathways and maintained gene expression patterns normally decreased by castration. Phosphoproteome profiling revealed the transcription factor FOXJ2 as a novel NEK6 substrate, with FOXJ2 phosphorylation associated with increased expression of newly identified NEK6 transcriptional targets. Overall, our studies establish NEK6 signaling as a central mechanism mediating castration-resistant prostate cancer. Cancer Res; 77(3); 753–65. ©2016 AACR.

Epithelial-mesenchymal transition (EMT) is an essential developmental program and is often reactivated during tumor initiation and progression. EMT is a reversible reprogramming of the cells. It not only promotes cell morphology alterations, but also provokes a profound cell state change in multiple regulatory circuits in global cell signaling, transcriptional and post-transcriptional modifications. Among all breast cancer subtypes, basal-like breast cancer displayed a high degree of mesenchymal and stem-like cell properties. To systematically interrogate the modulators of epithelial-to-mesenchymal transition, we performed a genome-scale ORF screen in human mammary epithelial cells. We used CD44 cell surface protein as a marker for cells reside in mesenchymal and stem-like cell state. CD44 low cells were presorted and induced with a barcoded genome-scale ORF library. After seven days of propagation, cells converted to CD44 high state were sorted by flow cytometry, and the barcodes enriched in the CD44 high population compared to the cells in the CD44 low population were evaluated with next-generation sequencing. Interestingly, 68 genes scored 3 standard deviations above the mean, including transcriptional factors, RNA splicing factors, epigenetic regulators, kinases/phosphatases, spermatogenesis regulators and amide metabolic modifiers. Strikingly, gene ontology and gene set enrichment analysis showed that RNA splicing process was the most significantly enriched biological process. We employed six different assays for candidate validation: 1) Induction of CD44 cell surface markers; 2) Evaluation of the expression of EMT markers; 3) Test of the ability to form mammospheres; 4) Investigation of the expression during EMT induction; 5) Test of the necessity of these splicing factors for EMT and stem-like states; 6) Examination of the ability to promote tumor formation in vivo. After performing these assays, we discovered the splicing factors/RNA binding proteins, QKI and RBFOX1, are both necessary and sufficient to promote EMT and stem-like states. We subsequently investigated the downstream targets of these splicing factors by RNA-sequencing analysis. Further, using biochemical and genetic approaches, we characterized the functional roles of these splicing factors and their downstream targets. In summary, alternative pre-mRNA splicing plays a key role in the regulation of EMT. The molecular targets and mechanism identified in this study may aid in the development of new diagnostic and therapeutic approaches for breast tumors, especially for basal-like breast cancer. Citation Format: Ji Li, Peter Choi, Christine Chaffer, Katherine Labella, Jong Wook Kim, John Doench, Chao Dai, Andrew Giacomelli, Seav Huong Ly, Justin Hwang, Andrew Hong, Nina Ilic, Ole Gjoerup, Matthew Meyerson, Angela Brooks, Robert Weinberg, William Hahn. Genome-scale ORF screening to identify modulators of the epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A36.

KRAS is the most commonly mutated oncogenes and is a major driver of tumor initiation and progression. Understanding the functional consequences of cancer-associated KRAS variants may have important clinical implications. For example, KRAS mutation status defines those that are likely to respond to EGFR-directed therapy in KRAS-mutant metastatic colorectal cancer. A compendium of all possible oncogenic KRAS alleles would serve as a roadmap for future therapeutic strategies directed at KRAS itself or downstream signaling effectors. Comprehensive mutagenesis of KRAS may also elucidate structure-function relationships that reveal novel biochemical properties that may be exploited for therapeutic gain. We performed saturation mutagenesis of both a wild-type (WT) and a G12D mutant form of KRAS cDNA and generated lentiviral expression libraries of 3,553 and 3,534 single amino acid substitution mutants of each backbone. We utilized these WT and G12D mutagenesis libraries for functional genetic screening to identify gain- and loss-of-function missense variants that alter critical oncogenic properties of KRAS. First, we sought to comprehensively identify all possible oncogenic missense mutations in KRAS that mediate oncogenic transformation. We stably transduced the WT library into immortalized human epithelial cells and evaluated growth in low attachment (GILA), an assay that is highly correlated with in vivo tumor formation. We identified all previously known hotspot oncogenic alleles of KRAS as well as many functionally relevant alleles that are also discovered at lower frequency in human tumors. Moreover, we also discovered a group of transforming KRAS variants that have not been well described in human tumors, thus revealing potentially novel activating mechanisms for oncogenic KRAS. In parallel, we utilized the G12D mutagenesis library to perform second-site suppressor screening to identify loss-of-function single amino acid changes that abrogate the transforming ability of oncogenic KRAS. We performed positive-selection screening in primary cell lines for variants that enable bypass of oncogene-induced senescence. Additionally, we conducted a negative-selection screen with the G12D library in a KRAS-dependent cancer cell line with inducible suppression of endogenous KRAS, thus identifying all possible second-site mutations that abolish KRAS-driven signaling necessary for maintenance of cellular proliferation and viability. Structure-function analysis of these data may reveal novel patterns of amino-acid changes that result in inactivation of oncogenic KRAS. In summary, this comprehensive dictionary of gain- and loss-of-function KRAS mutants will facilitate understanding of clinically important mutations and also yield novel insights into structure-function relationships that may improve our understanding of the KRAS oncogene. Citation Format: Eejung Kim, Seav Huong Ly, Nicole S. Persky, Belinda Wang, Xiaoping Yang, Federica Piccioni, Katherine Labella, Mihir Doshi, Robert E. Lintner, Cong Zhu, Scott Steelman, David E. Root, Cory M. Johannessen, Alex B. Burgin, Laura E. MacConaill, William C. Hahn, Andrew J. Aguirre. Saturation mutagenesis of KRAS reveals the functional landscape of missense variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 394. doi:10.1158/1538-7445.AM2017-394

Cells residing in mesenchymal state are often associated with stem cell properties. The phenotypic changes from epithelial to mesenchymal cell state, or from non-stem-like to stem-like cell state contribute to tumor heterogeneity and play important roles in tumor initiation, progression and metastasis. To systematically interrogate the modulators of epithelial-to-mesenchymal transition, we performed a genome-scale ORF screen to identify regulators of mesenchymal and stem-like cell states using a barcoded human ORFome expression library in human mammary epithelial cells. In the screen, we used flow cytometric analysis of the CD44 cell surface marker and identified 68 ORFs that can switch cells from CD44 low state to CD44 high state in 7 days. Among these genes, the RNA splicing factors were highly enriched as analyzed by GO terms and Gene set enrichment analysis (GSEA).We employed six different assays for candidate validation: 1) Induction of CD44 cell surface markers; 2) Evaluation of the expression of EMT markers; 3) Test of the ability to form mammospheres; 4) Investigation of the expression during EMT induction; 5) Test of the necessity of these splicing factors for EMT and stem-like states; 6) Examination of the ability to promote tumor formation in vivo. We discovered that QKI and RBFOX1 were both necessary and sufficient to promote EMT and stem-like states. MBNL1, MBNL2 and CELF4 were sufficient to induce some mesenchymal markers. We further investigated the downstream targets of these splicing factors by RNA-sequencing analysis. We found that QKI and RBFOX1 regulated the alternative splicing of genes in 5 functional modules: 1) Cell motility and ECM/cytoskeleton organization; 2) Stem cell fate determination; 3) Oncogenic signaling; 4) Epigenetic targets; 5) cell polarity. Strikingly, using molecular and biochemical assays, we found that QKI and RBFOX1/2 interacted and cooperatively regulated the alternative splicing of a large number of overlapping transcripts, including Filamin B (FLNB). QKI and RBFOX1 induced a shorter isoform via exon skipping. which plays a key functional role in the regulation of EMT. Importantly, the expression of QKI, RBFOX1/2 and the short isoform of FLNB are elevated in basal B type of breast cancer cell lines and in basal-like breast cancer patient samples, the subtype of breast cancer that displays higher degree of mesenchymal and stem-like traits. In conclusion, alternative RNA splicing plays a key role in the regulation of EMT and stem-like cell states. QKI and RBFOX1/2 are both necessary and sufficient to promote EMT and stem-like traits. Alternative splicing of FLNB controlled by QKI and RBFOX1/2 is one of the key downstream targets that regulates EMT. Thus, the molecular targets and mechanism identified in this study may aid in the development of new diagnostic and therapeutic approaches for breast tumors, especially for basal-like breast cancer. Citation Format: Ji Li, Peter Choi, Christine Chaffer, Katherine Labella, Jong Wook Kim, John Doench, Chao Dai, Andrew Giacomelli, Seav Huong Ly, Justin Hwang, Andrew Hong, Nina Ilic, Ole Gjoerup, Matthew Meyerson, Angela Brooks, Robert Weinberg, William Hahn. A genome-scale ORF screen reveals an alternative splicing program that regulates mesenchymal and stem-like cell states in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5020. doi:10.1158/1538-7445.AM2017-5020