Tirtha K. Das

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.

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 fields 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. Developmental Dynamics 241:107–118, 2012.

Sin3a acts through a multi-gene module to regulate invasion in Drosophila and human tumors

Chromatin remodeling proteins regulate multiple aspects of cell homeostasis, making them ideal candidates for misregulation in transformed cells. Here, we explore Sin3A, a member of the Sin3 family of proteins linked to tumorigenesis that are thought to regulate gene expression through their role as histone deacetylases (HDACs). We identified Drosophila Sin3a as an important mediator of oncogenic Ret receptor in a fly model of Multiple Endocrine Neoplasia Type 2. Reducing Drosophila Sin3a activity led to metastasis-like behavior and, in the presence of Diap1, secondary tumors distant from the site of origin. Genetic and Chip-Seq analyses identified previously undescribed Sin3a targets including genes involved in cell motility and actin dynamics, as well as signaling pathways including Src, Jnk and Rho. A key Sin3a oncogenic target, PP1B, regulates stability of β-Catenin/Armadillo: the outcome is to oppose T-cell factor (TCF) function and Wg/Wnt pathway signaling in both fly and mammalian cancer cells. Reducing Sin3A strongly increased the invasive behavior of A549 human lung adenocarcinoma cells. We show that Sin3A is downregulated in a variety of human tumors and that Src, JNK, RhoA and PP1B/β-Catenin are regulated in a manner analogous to our Drosophila models. Our data suggest that Sin3A influences a specific step of tumorigenesis by regulating a module of genes involved in cell invasion. Tumor progression may commonly rely on such ‘modules of invasion’ under the control of broad transcriptional regulators.

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.

Centrosomal kinase Nek2 cooperates with oncogenic pathways to promote metastasis

Centrosomal kinase Nek2 is overexpressed in different cancers, yet how it contributes toward tumorigenesis remains poorly understood. dNek2 overexpression in a Drosophila melanogaster model led to upregulation of Drosophila Wnt ortholog wingless (Wg), and alteration of cell migration markers—Rho1, Rac1 and E-cadherin (Ecad)—resulting in changes in cell shape and tissue morphogenesis. dNek2 overexpression cooperated with receptor tyrosine kinase and mitogen-activated protein kinase signaling to upregulate activated Akt, Diap1, Mmp1 and Wg protein to promote local invasion, distant seeding and metastasis. In tumor cell injection assays, dNek2 cooperated with Ras and Src signaling to promote aggressive colonization of tumors into different adult fly tissues. Inhibition of the PI3K pathway suppressed the cooperation of dNek2 with other growth pathways. Consistent with our fly studies, overexpression of human Nek2 in A549 lung adenocarcinoma and HEK293T cells led to activation of the Akt pathway and increase in β-catenin protein levels. Our computational approach identified a class of Nek2-inhibitory compounds and a novel drug-like pharmacophore that reversed the Nek2 overexpression phenotypes in flies and human cells. Our finding posits a novel role for Nek2 in promoting metastasis in addition to its currently defined role in promoting chromosomal instability. It provides a rationale for the selective advantage of centrosome amplification in cancer.

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.

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.

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.

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.

Abstract 1826: Anti-tumorigenic effects by targeted functional disruption of the Sin3 PAH-2 domain

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The Sin3 A/B adapter proteins function as structural scaffolds for repressor/activator complexes that regulate transcription through the specific association with histone modifying enzymes and a number of transcription factors. Sin3 contains four paired amphipathic α-helices (PAH domains). We have reported earlier that targeted disruption of the PAH2 domain with a SID (Sin3 Interaction Domain) peptide decoy in triple negative (TN) breast cancer cells leads to cytoskeletal reorganization, loss of anchorage independent growth and 3D invasive morphology and decreased cell adhesion and invasion. There is epigenetic reprogramming of silenced genes such as CDH1, ESR1 and RARA which are re-expressed and together contribute to a SID decoy induced switch from basal to a more differentiated luminal phenotype (Farias, et. al., PNAS, 2010, 107:11811-6). Computerized screening coupled with such assays as Duolink, GST pull downs and mammalian two hybrid identified small molecule inhibitors (SMI) that mimic the effects of the SID decoy peptide. SMI inhibit cellular invasion at nanomolar range and in in vivo mouse myc TN breast cancer prolong latency, decrease local invasion and metastasis. Tumors recovered showed evidence of re-expression of E-cadherin and estrogen receptor. Early effects of SID decoy in TN breast cancer cells include inhibition of invasion that is associated with a significant decrease in Src phosphorylation within 2-4hr of treatment. Recovery of phosphorylation after SID decoy washout is coupled with recovered invasion at 24hr. These effects occurred prior to measurable increase in E-cadherin expression, suggesting a non-transcriptional effect. In Drosophila larval breast cancer models with activated Src/Ras, overexpression of SID inhibited (60%) tumor growth in the eye imaginal disc, and was eradicated by the addition of a MEK inhibitor (AZD-6244), indicating a strong synergy between SID and AZD-6244. In the adult fly SID expression greatly inhibited RetMEN2B induced eye tumors (90%). These data demonstrate that SID decoys have a potential to be effective agents in the treatment of TN breast cancer by promoting basal phenotype reversal, inhibiting invasion and metastasis, and could be synergistic with specific inhibitors of signal transduction targets. Moreover, SID decoys can overcome profound oncogenic stimulus such as Ras, Src and Ret suggesting that they have a potential greater role than just in the treatment of TN breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1826. doi:1538-7445.AM2012-1826