Functional exploration of copy number alterations in a Drosophila model of triple-negative breast cancer.

Accounting for 10-20% of breast cancer cases, triple-negative breast cancer (TNBC) is associated with a disproportionate number of breast cancer deaths. One challenge in studying TNBC is its genomic profile: with the exception of TP53 loss, most breast cancer tumors are characterized by a high number of copy number alterations (CNAs), making modeling the disease in whole animals challenging. We computationally analyzed 186 CNA regions previously identified in breast cancer tumors to rank genes within each region by likelihood of acting as a tumor driver. We then used a Drosophila p53-Myc TNBC model to identify 48 genes as functional drivers. To demonstrate the utility of this functional database, we established six 3-hit models; altering candidate genes led to increased aspects of transformation as well as resistance to the chemotherapeutic drug fluorouracil. Our work provides a functional database of CNA-associated TNBC drivers, and a template for an integrated computational/whole-animal approach to identify functional drivers of transformation and drug resistance within CNAs in other tumor types.

The seventh international RASopathies symposium: Pathways to a cure-expanding knowledge, enhancing research, and therapeutic discovery.

RASopathies are a group of genetic disorders that are caused by genes that affect the canonical Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Despite tremendous progress in understanding the molecular consequences of these genetic anomalies, little movement has been made in translating these findings to the clinic. This year, the seventh International RASopathies Symposium focused on expanding the research knowledge that we have gained over the years to enhance new discoveries in the field, ones that we hope can lead to effective therapeutic treatments. Indeed, for the first time, research efforts are finally being translated to the clinic, with compassionate use of Ras/MAPK pathway inhibitors for the treatment of RASopathies. This biannual meeting, organized by the RASopathies Network, brought together basic scientists, clinicians, clinician scientists, patients, advocates, and their families, as well as representatives from pharmaceutical companies and the National Institutes of Health. A history of RASopathy gene discovery, identification of new disease genes, and the latest research, both at the bench and in the clinic, were discussed.

Nek2 Kinase Signaling in Malaria, Bone, Immune and Kidney Disorders to Metastatic Cancers and Drug Resistance: Progress on Nek2 Inhibitor Development.

Cell cycle kinases represent an important component of the cell machinery that controls signal transduction involved in cell proliferation, growth, and differentiation. Nek2 is a mitotic Ser/Thr kinase that localizes predominantly to centrosomes and kinetochores and orchestrates centrosome disjunction and faithful chromosomal segregation. Its activity is tightly regulated during the cell cycle with the help of other kinases and phosphatases and via proteasomal degradation. Increased levels of Nek2 kinase can promote centrosome amplification (CA), mitotic defects, chromosome instability (CIN), tumor growth, and cancer metastasis. While it remains a highly attractive target for the development of anti-cancer therapeutics, several new roles of the Nek2 enzyme have recently emerged: these include drug resistance, bone, ciliopathies, immune and kidney diseases, and parasitic diseases such as malaria. Therefore, Nek2 is at the interface of multiple cellular processes and can influence numerous cellular signaling networks. Herein, we provide a critical overview of Nek2 kinase biology and discuss the signaling roles it plays in both normal and diseased human physiology. While the majority of research efforts over the last two decades have focused on the roles of Nek2 kinase in tumor development and cancer metastasis, the signaling mechanisms involving the key players associated with several other notable human diseases are highlighted here. We summarize the efforts made so far to develop Nek2 inhibitory small molecules, illustrate their action modalities, and provide our opinion on the future of Nek2-targeted therapeutics. It is anticipated that the functional inhibition of Nek2 kinase will be a key strategy going forward in drug development, with applications across multiple human diseases.

Crowdsourced identification of multi-target kinase inhibitors for RET- and TAU- based disease: The Multi-Targeting Drug DREAM Challenge.

A continuing challenge in modern medicine is the identification of safer and more efficacious drugs. Precision therapeutics, which have one molecular target, have been long promised to be safer and more effective than traditional therapies. This approach has proven to be challenging for multiple reasons including lack of efficacy, rapidly acquired drug resistance, and narrow patient eligibility criteria. An alternative approach is the development of drugs that address the overall disease network by targeting multiple biological targets ('polypharmacology'). Rational development of these molecules will require improved methods for predicting single chemical structures that target multiple drug targets. To address this need, we developed the Multi-Targeting Drug DREAM Challenge, in which we challenged participants to predict single chemical entities that target pro-targets but avoid anti-targets for two unrelated diseases: RET-based tumors and a common form of inherited Tauopathy. Here, we report the results of this DREAM Challenge and the development of two neural network-based machine learning approaches that were applied to the challenge of rational polypharmacology. Together, these platforms provide a potentially useful first step towards developing lead therapeutic compounds that address disease complexity through rational polypharmacology.

Drosophila RASopathy models identify disease subtype differences and biomarkers of drug efficacy.

RASopathies represent a family of mostly autosomal dominant diseases that are caused by missense variants in the rat sarcoma viral oncogene/mitogen activated protein kinase (RAS/MAPK) pathway including KRAS, NRAS, BRAF, RAF1, and SHP2. These variants are associated with overlapping but distinct phenotypes that affect the heart, craniofacial, skeletal, lymphatic, and nervous systems. Here, we report an analysis of 13 Drosophila transgenic lines, each expressing a different human RASopathy isoform. Similar to their human counterparts, each Drosophila line displayed common aspects but also important differences including distinct signaling pathways such as the Hippo and SAPK/JNK signaling networks. We identified multiple classes of clinically relevant drugs-including statins and histone deacetylase inhibitors-that improved viability across most RASopathy lines; in contrast, several canonical RAS pathway inhibitors proved less broadly effective. Overall, our study compares and contrasts a large number of RASopathy-associated variants including their therapeutic responses.

Restraining Network Response to Targeted Cancer Therapies Improves Efficacy and Reduces Cellular Resistance.

A key tool of cancer therapy has been targeted inhibition of oncogene-addicted pathways. However, efficacy has been limited by progressive emergence of resistance as transformed cells adapt. Here, we use Drosophila to dissect response to targeted therapies. Treatment with a range of kinase inhibitors led to hyperactivation of overall cellular networks, resulting in emergent resistance and expression of stem cell markers, including Sox2. Genetic and drug screens revealed that inhibitors of histone deacetylases, proteasome, and Hsp90 family of proteins restrained this network hyperactivation. These "network brake" cocktails, used as adjuncts, prevented emergent resistance and promoted cell death at subtherapeutic doses. Our results highlight a general response of cells, transformed and normal, to targeted therapies that leads to resistance and toxicity. Pairing targeted therapeutics with subtherapeutic doses of broad-acting "network brake" drugs may provide a means of extending therapeutic utility while reducing whole body toxicity.Significance: These findings with a strong therapeutic potential provide an innovative approach of identifying effective combination treatments for cancer. Cancer Res; 78(15); 4344-59. ©2018 AACR.

Non-mammalian models of multiple endocrine neoplasia type 2.

Twenty-five years ago, RET was identified as the primary driver of multiple endocrine neoplasia type 2 (MEN2) syndrome. MEN2 is characterized by several transformation events including pheochromocytoma, parathyroid adenoma and, especially penetrant, medullary thyroid carcinoma (MTC). Overall, MTC is a rare but aggressive type of thyroid cancer for which no effective treatment currently exists. Surgery, radiation, radioisotope treatment and chemotherapeutics have all shown limited success, and none of these approaches have proven durable in advanced disease. Non-mammalian models that incorporate the oncogenic RET isoforms associated with MEN2 and other RET-associated diseases have been useful in delineating mechanisms underlying disease progression. These models have also identified novel targeted therapies as single agents and as combinations. These studies highlight the importance of modeling disease in the context of the whole animal, accounting for the complex interplay between tumor and normal cells in controlling disease progression as well as response to therapy. With convenient access to whole genome sequencing data from expanded thyroid cancer patient cohorts, non-mammalian models will become more complex, sophisticated and continue to complement future mammalian studies. In this review, we explore the contributions of non-mammalian models to our understanding of thyroid cancer including MTC, with a focus on Danio rerio and Drosophila melanogaster (fish and fly) models.

KIF5B-RET Oncoprotein Signals through a Multi-kinase Signaling Hub.

Gene fusions are increasingly recognized as important cancer drivers. The KIF5B-RET gene has been identified as a primary driver in a subset of lung adenocarcinomas. Targeting human KIF5B-RET to epithelia in Drosophila directed multiple aspects of transformation, including hyperproliferation, epithelial-to-mesenchymal transition, invasion, and extension of striking invadopodia-like processes. The KIF5B-RET-transformed human bronchial cell line showed similar aspects of transformation, including invadopodia-like processes. Through a combination of genetic and biochemical studies, we demonstrate that the kinesin and kinase domains of KIF5B-RET act together to establish an emergent microtubule and RAB-vesicle-dependent RET-SRC-EGFR-FGFR signaling hub. We demonstrate that drugs designed to inhibit RET alone work poorly in KIF5B-RET-transformed cells. However, combining the RET inhibitor sorafenib with drugs that target EGFR, microtubules, or FGFR led to strong efficacy in both Drosophila and human cell line KIF5B-RET models. This work demonstrates the utility of exploring the full biology of fusions to identify rational therapeutic strategies.

A Drosophila approach to thyroid cancer therapeutics.

Thyroid neoplasias represent among the fastest growing cancers. While surgery has become the treatment of choice for most thyroid tumors, many require chemotherapy. In this review, we examine the contributions of work in the fruit fly Drosophila toward multiple endocrine neoplasia type 2 (MEN2), a Ret-based disease to which recent Drosophila models have proven useful both for understanding disease mechanism as well as helping identify new generation therapeutics.

γ-H2AX kinetics as a novel approach to high content screening for small molecule radiosensitizers.

BACKGROUND: Persistence of γ-H2AX after ionizing radiation (IR) or drug therapy is a robust reporter of unrepaired DNA double strand breaks in treated cells. METHODS: DU-145 prostate cancer cells were treated with a chemical library ±IR and assayed for persistence of γ-H2AX using an automated 96-well immunocytochemistry assay at 4 hours after treatment. Hits that resulted in persistence of γ-H2AX foci were tested for effects on cell survival. The molecular targets of hits were validated by molecular, genetic and biochemical assays and in vivo activity was tested in a validated Drosophila cancer model. RESULTS: We identified 2 compounds, MS0019266 and MS0017509, which markedly increased persistence of γ-H2AX, apoptosis and radiosensitization in DU-145 cells. Chemical evaluation demonstrated that both compounds exhibited structurally similar and biochemical assays confirmed that these compounds inhibit ribonucleotide reductase. DNA microarray analysis and immunoblotting demonstrates that MS0019266 significantly decreased polo-like kinase 1 gene and protein expression. MS0019266 demonstrated in vivo antitumor activity without significant whole organism toxicity. CONCLUSIONS: MS0019266 and MS0017509 are promising compounds that may be candidates for further development as radiosensitizing compounds as inhibitors of ribonucleotide reductase.

Chemical genetic discovery of targets and anti-targets for cancer polypharmacology.

The complexity of cancer has led to recent interest in polypharmacological approaches for developing kinase-inhibitor drugs; however, optimal kinase-inhibition profiles remain difficult to predict. Using a Ret-kinase-driven Drosophila model of multiple endocrine neoplasia type 2 and kinome-wide drug profiling, here we identify that AD57 rescues oncogenic Ret-induced lethality, whereas related Ret inhibitors imparted reduced efficacy and enhanced toxicity. Drosophila genetics and compound profiling defined three pathways accounting for the mechanistic basis of efficacy and dose-limiting toxicity. Inhibition of Ret plus Raf, Src and S6K was required for optimal animal survival, whereas inhibition of the 'anti-target' Tor led to toxicity owing to release of negative feedback. Rational synthetic tailoring to eliminate Tor binding afforded AD80 and AD81, compounds featuring balanced pathway inhibition, improved efficacy and low toxicity in Drosophila and mammalian multiple endocrine neoplasia type 2 models. Combining kinase-focused chemistry, kinome-wide profiling and Drosophila genetics provides a powerful systems pharmacology approach towards developing compounds with a maximal therapeutic index.

Design, synthesis, and evaluation of 2-(arylsulfonyl)oxiranes as cell-permeable covalent inhibitors of protein tyrosine phosphatases.

A structure-based design approach has been applied to develop 2-(arylsulfonyl)oxiranes as potential covalent inhibitors of protein tyrosine phosphatases. A detailed kinetic analysis of inactivation by these covalent inhibitors reveals that this class of compounds inhibits a panel of protein tyrosine phosphatases in a time- and dose-dependent manner, consistent with the covalent modification of the enzyme active site. An inactivation experiment in the presence of sodium arsenate, a known competitive inhibitor of protein tyrosine phosphatase, indicated that these inhibitors were active site bound. This finding is consistent with the mass spectrometric analysis of the covalently modified protein tyrosine phosphatase enzyme. Additional experiments indicated that these compounds remained inert toward other classes of arylphosphate-hydrolyzing enzymes, and alkaline and acid phosphatases. Cell-based experiments with human A549 lung cancer cell lines indicated that 2-(phenylsulfonyl)oxirane (1) caused an increase in intracellular pTyr levels in a dose-dependent manner thereby suggesting its cell-permeable nature. Taken together, the newly identified 2-(arylsulfonyl)oxiranyl moiety could serve as a novel chemotype for the development of activity-based probes and therapeutic agents against protein tyrosine phosphatase superfamily of enzymes.

Drosophila cancer models.

Cancer is driven by complex genetic and cellular mechanisms. Recently, the Drosophila community has become increasingly interested in exploring cancer issues. The Drosophila field has made seminal contributions to many of the mechanisms that are fundamental to the cancer process; several of these mechanisms have already been validated in vertebrates. Less well known are the Drosophila field's early direct contributions to the cancer field: some of the earliest tumor suppressors were identified in flies. In this review, we identify major contributions that Drosophila studies have made toward dissecting the pathways and mechanisms underlying tumor progression. We also highlight areas, such as drug discovery, where we expect Drosophila studies to make a major scientific impact in the future.

Drosophila as a novel therapeutic discovery tool for thyroid cancer.

BACKGROUND: Multiple endocrine neoplasia type II (MEN2) is a rare but aggressive cancer for which no effective treatment currently exists. A Drosophila model was developed to identify novel genetic modifier loci of oncogenic RET, as well as to provide a whole animal system to rapidly identify compounds that suppressed RET-dependent MEN2. ZD6474 (Vandetanib), currently in phase III trials, suppressed tumorigenesis in MEN2 model flies, demonstrating for the first time the effectiveness of a Drosophila-based whole animal model for identifying therapeutically useful compounds. SUMMARY: Clinical data suggest that drug mono-therapy for MEN2 and other cancers typically yield only moderate benefits as patients develop drug resistance and suffer from drug-induced pathway feedback. Combinations of drugs that target different nodes of the oncogenic pathway are an effective way to prevent resistance as well as feedback. Identifying the optimal drug-dose combinations for therapy poses a significant challenge in existing mouse models. Fly models offer a means to quickly and effectively identify drug combinations that are well tolerated and potently suppress the MEN2 phenotype. This approach may also identify differences in therapeutic responses between the two subtypes of MEN2--MEN2A and MEN2B--providing additional therapeutic insights. CONCLUSIONS: Fly models have proven useful for identifying known drugs as well as novel compounds that, as single agents or in combinations, effectively suppress the MEN2 syndrome. These findings validate the use of fly models for both drug discovery as well as identification of useful drug combinations. In the future, rapid pairing of new genomic information with increasingly complex fly models will aid us in efforts to further tailor drug treatments toward personalized medicine.

A conserved F-box gene with unusual transcript localization.

Drosophila partner of paired ( ppa), which encodes an F-box protein that targets the Pax transcription factor Paired (Prd) for degradation, has the striking property that its mRNA is expressed in a striped pattern with a characteristic registration relative to the striped expression of prd in early embryos. Localized expression of F-box genes was not expected because F-box proteins generally have multiple substrates. We hypothesize that the patterned mRNA expression of Drosophila and zebrafish ppa homologs may reflect constraints resulting from the localized expression of their degradation substrates. To begin to test this idea, we wished to determine whether patterned mRNA expression is commonly observed among F-box genes, or whether it might be peculiar to ppa and its homologs, or even specific to Drosophila. We examined embryonic expression of all predicted F-box genes in Drosophila and found that mRNAs of 21 out of 23 predicted F-box genes are expressed uniformly in early Drosophila embryos, whereas ppa and CG4911 mRNAs are patterned, CG4911 being expressed at the positions of gastrulation folds. We also identified and tested expression of ppa in zebrafish, which has two highly conserved homologs, ppaA and ppaB, and found that both are expressed during embryogenesis and have enriched mRNA expression in regions including the neural tube, the head, and the fin buds. Despite being unusual in having localized transcripts, we found that the Drosophila and zebrafish homologs interact with the expected Drosophila components of the cellular degradation machinery - Skp1 (SkpA) and Rbx1 (Roc1a) - suggesting that the Ppa proteins are indeed functional F-box proteins. We conclude that patterning of ppa mRNA could reflect a constraint on ppa function that is not common among F-box genes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00427-002-0222-7.