Mechanistic Interrogation Research Articles

Identification of a Clinically Relevant Signature for Early Progression in KRAS-Driven Lung Adenocarcinoma.

Inducible genetically defined mouse models of cancer uniquely facilitate the investigation of early events in cancer progression, however, there are valid concerns about the ability of such models to faithfully recapitulate human disease. We developed an inducible mouse model of progressive lung adenocarcinoma (LuAd) that combines sporadic activation of oncogenic KRasG12D with modest overexpression of c-MYC (KM model). Histological examination revealed a highly reproducible spontaneous transition from low-grade adenocarcinoma to locally invasive adenocarcinoma within 6 weeks of oncogene activation. Laser-capture microdissection coupled with RNA-SEQ (ribonucleic acid sequencing) was employed to determine transcriptional changes associated with tumour progression. Upregulated genes were triaged for relevance to human LuAd using datasets from Oncomine and cBioportal. Selected genes were validated by RNAi screening in human lung cancer cell lines and examined for association with lung cancer patient overall survival using KMplot.com. Depletion of progression-associated genes resulted in pronounced viability and/or cell migration defects in human lung cancer cells. Progression-associated genes moreover exhibited strong associations with overall survival, specifically in human lung adenocarcinoma, but not in squamous cell carcinoma. The KM mouse model faithfully recapitulates key molecular events in human adenocarcinoma of the lung and is a useful tool for mechanistic interrogation of KRAS-driven LuAd progression.

Discovering metabolic disease gene interactions by correlated effects on cellular morphology

ObjectiveImpaired expansion of peripheral fat contributes to the pathogenesis of insulin resistance and Type 2 Diabetes (T2D). We aimed to identify novel disease–gene interactions during adipocyte differentiation. MethodsGenes in disease-associated loci for T2D, adiposity and insulin resistance were ranked according to expression in human adipocytes. The top 125 genes were ablated in human pre-adipocytes via CRISPR/CAS9 and the resulting cellular phenotypes quantified during adipocyte differentiation with high-content microscopy and automated image analysis. Morphometric measurements were extracted from all images and used to construct morphologic profiles for each gene. ResultsOver 107 morphometric measurements were obtained. Clustering of the morphologic profiles accross all genes revealed a group of 14 genes characterized by decreased lipid accumulation, and enriched for known lipodystrophy genes. For two lipodystrophy genes, BSCL2 and AGPAT2, sub-clusters with PLIN1 and CEBPA identifed by morphological similarity were validated by independent experiments as novel protein–protein and gene regulatory interactions. ConclusionsA morphometric approach in adipocytes can resolve multiple cellular mechanisms for metabolic disease loci; this approach enables mechanistic interrogation of the hundreds of metabolic disease loci whose function still remains unknown.

Genomic knockout of alms1 in zebrafish recapitulates Alström syndrome and provides insight into metabolic phenotypes.

Alström syndrome (OMIM #203800) is an autosomal recessive obesity ciliopathy caused by loss-of-function mutations in the ALMS1 gene. In addition to multi-organ dysfunction, such as cardiomyopathy, retinal degeneration and renal dysfunction, the disorder is characterized by high rates of obesity, insulin resistance and early-onset type 2 diabetes mellitus (T2DM). To investigate the underlying mechanisms of T2DM phenotypes, we generated a loss-of-function deletion of alms1 in the zebrafish. We demonstrate conservation of hallmark clinical characteristics alongside metabolic syndrome phenotypes, including a propensity for obesity and fatty livers, hyperinsulinemia and glucose response defects. Gene expression changes in β-cells isolated from alms1-/- mutants revealed changes consistent with insulin hypersecretion and glucose sensing failure, which were corroborated in cultured murine β-cells lacking Alms1. We also found evidence of defects in peripheral glucose uptake and concomitant hyperinsulinemia in the alms1-/- animals. We propose a model in which hyperinsulinemia is the primary and causative defect underlying generation of T2DM associated with alms1 deficiency. These observations support the alms1 loss-of-function zebrafish mutant as a monogenic model for mechanistic interrogation of T2DM phenotypes.

Type I Protein Arginine Methyltransferases Overexpression Promotes Transformation and Potentiates Her2/Neu-Driven Tumorigenesis.

Bao and colleagues demonstrate that type I protein arginine methyltransferases (PRMT) are directly involved in mammary gland transformation and tumor progression. Notably, several distinct phenotypes require further investigation such as PRMT1/CARM1-induced transformation, CARM1-mediated delay in tumorigenesis, and PRMTs potentiation of Her2-dependent tumors. The PRMT overexpression transgenic mouse models should encourage and facilitate further mechanistic interrogation and the development of PRMT-directed therapies.See related article by Bao et al., p. 21.

CRISPR-Cas9 genome engineering of primary CD4+ T cells for the interrogation of HIV-host factor interactions.

CRISPR-Cas9 gene-editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology for use in primary human CD4+ T cells to create a high-throughput platform for analyzing the role of host factors in HIV infection and pathogenesis. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated CD4+ T cells by nucleofection. These cells are then assayed for editing efficiency and expanded for use in downstream cellular, genetic, or protein-based assays. This platform supports the rapid, arrayed generation of multiple gene manipulations and is widely adaptable across culture conditions, infection protocols, and downstream applications. Here, we present detailed protocols for crRNP synthesis, primary T-cell culture, 96-well nucleofection, molecular validation, and HIV infection, and discuss additional considerations for guide and screen design, as well as crRNP multiplexing. Taken together, this procedure allows high-throughput identification and mechanistic interrogation of HIV host factors in primary CD4+ T cells by gene knockout, validation, and HIV spreading infection in as little as 2-3 weeks.

Longitudinal single-cell RNA sequencing of patient-derived primary cells reveals drug-induced infidelity in stem cell hierarchy

Chemo-resistance is one of the major causes of cancer-related deaths. Here we used single-cell transcriptomics to investigate divergent modes of chemo-resistance in tumor cells. We observed that higher degree of phenotypic intra-tumor heterogeneity (ITH) favors selection of pre-existing drug-resistant cells, whereas phenotypically homogeneous cells engage covert epigenetic mechanisms to trans-differentiate under drug-selection. This adaptation was driven by selection-induced gain of H3K27ac marks on bivalently poised resistance-associated chromatin, and therefore not expressed in the treatment-naïve setting. Mechanistic interrogation of this phenomenon revealed that drug-induced adaptation was acquired upon the loss of stem factor SOX2, and a concomitant gain of SOX9. Strikingly we observed an enrichment of SOX9 at drug-induced H3K27ac sites, suggesting that tumor evolution could be driven by stem cell-switch-mediated epigenetic plasticity. Importantly, JQ1 mediated inhibition of BRD4 could reverse drug-induced adaptation. These results provide mechanistic insights into the modes of therapy-induced cellular plasticity and underscore the use of epigenetic inhibitors in targeting tumor evolution.

The High Chemofidelity of Metal-Catalyzed Hydrogen Atom Transfer.

The implementation of any chemical reaction in a structurally complex setting ( King , S. M. J. Org. Chem. 2014 , 79 , 8937 ) confronts structurally defined barriers: steric environment, functional group reactivity, product instability, and through-bond electronics. However, there are also practical barriers. Late-stage reactions conducted on small quantities of material are run inevitably at lower than optimal concentrations. Access to late-stage material limits extensive optimization. Impurities from past reactions can interfere, especially with catalytic reactions. Therefore, chemical reactions on which one can rely at the front lines of a complex synthesis campaign emerge from the crucible of total synthesis as robust, dependable, and widely applied. Trost conceptualized "chemoselectivity" as a reagent's selective reaction of one functional group or reactive site in preference to others ( Trost , B. M. Science 1983 , 219 , 245 ). Chemoselectivity and functional group tolerance can be evaluated quickly using robustness screens ( Collins , K. D. Nat. Chem. 2013 , 5 , 597 ). A reaction may also be characterized by its "chemofidelity", that is, its reliable reaction with a functional group in any molecular context. For example, ketone reduction by an electride (dissolving metal conditions) exhibits high chemofidelity but low chemoselectivity: it usually works, but many other functional groups are reduced at similar rates. Conversely, alkene coordination chemistry effected by π Lewis acids can exhibit high chemoselectivity ( Trost , B. M. Science 1983 , 219 , 245 ) but low chemofidelity: it can be highly selective for alkenes but sensitive to the substitution pattern ( Larionov , E. Chem. Commun. 2014 , 50 , 9816 ). In contrast, alkenes undergo reliable, robust, and diverse hydrogen atom transfer reactions from metal hydrides to generate carbon-centered radicals. Although there are many potential applications of this chemistry, its functional group tolerance, high rates, and ease of execution have led to its rapid deployment in complex synthesis campaigns. Its success derives from high chemofidelity, that is, its dependable reactivity in many molecular environments and with many alkene substitution patterns. Metal hydride H atom transfer (MHAT) reactions convert diverse, simple building blocks to more stereochemically and functionally dense products ( Crossley , S. W. M. Chem. Rev. 2016 , 116 , 8912 ). When hydrogen is returned to the metal, MHAT can be considered the radical equivalent of Brønsted acid catalysis-itself a broad reactivity paradigm. This Account summarizes our group's contributions to method development, reagent discovery, and mechanistic interrogation. Our earliest contribution to this area-a stepwise hydrogenation with high chemoselectivity and high chemofidelity-has found application to many problems. More recently, we reported the first examples of dual-catalytic cross-couplings that rely on the merger of MHAT cycles and nickel catalysis. With time, we anticipate that MHAT will become a staple of chemical synthesis.

Mouse Models for Cancer Immunotherapy Research.

Immunotherapy has revolutionized cancer therapy, largely attributed to the success of immune-checkpoint blockade. However, there are subsets of patients across multiple cancers who have not shown robust responses to these agents. A major impediment to progress in the field is the availability of faithful mouse models that recapitulate the complexity of human malignancy and immune contexture within the tumor microenvironment. These models are urgently needed across all malignancies to interrogate and predict antitumor immune responses and therapeutic efficacy in clinical trials. Herein, we seek to review pros and cons of different cancer mouse models, and how they can be used as platforms to predict efficacy and resistance to cancer immunotherapies.Significance: Although immunotherapy has shown substantial benefit in the treatment of a variety of malignancies, a key hurdle toward the advancement of these therapies is the availability of immunocompetent preclinical mouse models that recapitulate human disease. Here, we review the evolution of preclinical mouse models and their utility as coclinical platforms for mechanistic interrogation of cancer immunotherapies. Cancer Discov; 8(11); 1358-65. ©2018 AACR.

Mechanistic Interrogation of Co/Ni-Dual Catalyzed Hydroarylation.

Cobalt/nickel-dual catalyzed hydroarylation of terminal olefins with iodoarenes builds complexity from readily available starting materials, with a high preference for the Markovnikov (branched) product. Here, we advance a mechanistic model of this reaction through the use of reaction progress kinetic analysis (RPKA), radical clock experiments, and stoichiometric studies. Through exclusion of competing hypotheses, we conclude that the reaction proceeds through an unprecedented alkylcobalt to nickel direct transmetalation. Demonstration of catalytic alkene prefunctionalization, via spectroscopic observation of an organocobalt species, distinguishes this Csp2-Csp3 cross-coupling method from a conventional transmetalation process, which employs a stoichiometric organometallic nucleophile, and from a bimetallic oxidative addition of an organohalide across nickel, described by radical scission and subsequent alkyl radical capture at a second nickel center. A refined understanding of the reaction leads to an optimized hydroarylation procedure that excludes exogenous oxidant, demonstrating that the transmetalation is net redox neutral. Catalytic alkene prefunctionalization by cobalt and engagement with nickel catalytic cycles through direct transmetalation provides a new platform to merge these two rich areas of chemistry in preparatively useful ways.

Abstract B018: A high-throughput screen identifies HSP90 inhibitors as potent therapeutics across multiple clinically representative organoid models of advanced prostate cancer

Abstract Androgen-deprivation therapy (ADT) remains the gold-standard therapy for prostate cancer (PrCa), and although ADT is initially effective, most men progress to castrate-resistant prostate cancer (CRPC) within 2-3 years. Advanced CRPC is challenging to treat because intrinsic tumor heterogeneity and phenotypic plasticity engender short-lived responses and underlie resistance to conventional therapies. Combined PTEN/TP53 alterations represent a major genotype of advanced CRPC (25-30%) and are associated with poor clinical outcomes. Established PrCa cell lines do not accurately represent the heterogeneity of advanced CRPC, and therefore, nonbiased pharmacogenomics screens have not been done. The development of clinically representative, tractable models suitable for high-throughput target identification and validation is crucial for advancing novel CRPC therapies to the clinic. A comprehensive nonbiased high-throughput screen performed on seven cell lines derived from a genetically engineered mouse model (GEMM) of Pten/Tp53 null PrCa identified strongly active compounds, including inhibitors of PI3K/AKT/mTOR signaling, the proteasome, cell cycle regulatory proteins, heat shock proteins, DNA repair signaling, NFKB signaling, MAPK signaling, and several types of epigenetic modifiers. HSP90 inhibitors were one of the most efficacious classes of compounds in the screen, and ganetespib, a clinically used second-generation HSP90 inhibitor with a favorable safety profile, was the most potent. Although HSP90 inhibitors have yet to be successful as single agents, they have not been thoroughly investigated in clinically representative models of advanced PrCa and have shown potential as “network drugs,” prompting our investigations into their utility in polytherapy. We first validated ganetespib as a single agent, where it displayed strong activity against several GEMM-derived and LuCaP PDX-derived organoid models encompassing genotypic, phenotypic, and lineage heterogeneity. These 10 novel LuCaP PDX-derived organoids are representative of the numerous categories of CRPC, including adenocarcinomas with wild-type AR, adenocarcinomas with altered AR, adenocarcinoma with neuroendocrine features, and neuroendocrine disease. Single-agent ganetespib was also strongly inhibitory in vivo, decreasing growth of Pten/Tp53 null endogenous GEMM tumors as well as a human PDX tumor. Mechanistic interrogation of cell lines, organoids, and tumors exposed to ganetespib revealed inhibition of targets from several inter-related networks including AR and pAKT, two central and mutually compensatory growth and survival pathways for PrCa. The efficacy of ganetespib against a diverse group of CRPC organoids and the simultaneous inhibition of PrCa survival signaling suggested it may work well in combination. We performed a proof-of-principle high-throughput matrix screen on organoids derived from a Pten/Tp53 null GEMM and identified docetaxel and etoposide to be synergistic when combined with ganetespib. Preclinical in vivo studies to validate these findings are ongoing. In all, comprehensive data from multiple near-patient models suggest novel contexts for second-generation HSP90-directed intervention against a variety of CRPC genotypes and phenotypes and expand upon the potential of HSP90 inhibitors to simultaneously inhibit oncogenic signaling and compensatory resistance mechanisms. Citation Format: Keith H. Jansson, John B. Tucker, Lauren E. Stahl, John K. Simmons, Caitlyn Fuller, Michael L. Beshiri, Supreet Agarwal, Yasmine Abbey, Lei Fang, Paul G. Hynes, Alilin Aian Neil, Jacob Cawley, Ross Lake, Crystal Tran, Caitlin M. Tice, JuanJuan Yin, Xiahu Zhang, Rajarshi Guha, Shelley Hoover, R. Mark Simpson, Holly Nguyen, Eva Corey, Craig J. Thomas, David Proia, Kathleen Kelly. A high-throughput screen identifies HSP90 inhibitors as potent therapeutics across multiple clinically representative organoid models of advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B018.

Redox-Neutral Photocatalytic Cyclopropanation via Radical/Polar Crossover.

A benchtop stable, bifunctional reagent for the redox-neutral cyclopropanation of olefins has been developed. Triethylammonium bis(catecholato)iodomethylsilicate can be readily prepared on multigram scale. Using this reagent in combination with an organic photocatalyst and visible light, cyclopropanation of an array of olefins, including trifluoromethyl- and pinacolatoboryl-substituted alkenes, can be accomplished in a matter of hours. The reaction is highly tolerant of traditionally reactive functional groups (carboxylic acids, basic heterocycles, alkyl halides, etc.) and permits the chemoselective cyclopropanation of polyolefinated compounds. Mechanistic interrogation revealed that the reaction proceeds via a rapid anionic 3- exo- tet ring closure, a pathway consistent with experimental and computational data.

Mechanistic interrogation of the entry‐ and exit‐channel arms of eIF3

Eukaryotic translation initiation factor 3 (eIF3) is a pivotal player in the recruitment of mRNA to the translation pre‐initiation complex (PIC). Biochemical and genetic studies have identified interactions of eIF3 with the PIC near the mRNA entry and exit channels of the ribosome; structural studies have revealed that eIF3 binds the solvent face of the small (40S) ribosomal subunit, projecting arms into both mRNA channels and ultimately encircling the PIC. We recently probed the effects of a library of S. cerevisiae eIF3 functional variants using an in‐vitro reconstituted system. These experiments demonstrated that mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment of a native yeast mRNA. Using a series of model mRNAs to control the amount of nucleotide sequence in either the mRNA entry or exit channel, we further revealed a critical role for eIF3 in stabilizing mRNA at the exit channel. Together, these experiments are consistent with the entry‐ and exit‐channel arms of eIF3 playing distinct roles during PIC formation, mRNA recruitment, and scanning. We have also investigated the collaboration of eIF3 with Rps3/uS3 in stabilizing mRNA within the mRNA entry channel. We found that Rps3/uS3 residues previously implicated in the helicase activity of the elongating ribosome stabilize mRNA binding within ribosomal entry channel and enhance the effects of eIF3 on mRNA binding in both channels. More recently, we have observed that eIF3i and eIF3g contribute to the PIC‐dependent enhancement of eIF4A ATPase activity, which recent work suggests may function to remodel the mRNA entry channel. Our current work is focused on dissecting the interactions between eIF3, the PIC, and other initiation factors in both the mRNA entry and exit channels, as well as on the inter‐subunit face of the PIC, using both in‐vitro and next‐generation sequencing approaches.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A stochastic and dynamical view of pluripotency in mouse embryonic stem cells.

Pluripotent embryonic stem cells are of paramount importance for biomedical sciences because of their innate ability for self-renewal and differentiation into all major cell lines. The fateful decision to exit or remain in the pluripotent state is regulated by complex genetic regulatory networks. The rapid growth of single-cell sequencing data has greatly stimulated applications of statistical and machine learning methods for inferring topologies of pluripotency regulating genetic networks. The inferred network topologies, however, often only encode Boolean information while remaining silent about the roles of dynamics and molecular stochasticity inherent in gene expression. Herein we develop a framework for systematically extending Boolean-level network topologies into higher resolution models of networks which explicitly account for the promoter architectures and gene state switching dynamics. We show the framework to be useful for disentangling the various contributions that gene switching, external signaling, and network topology make to the global heterogeneity and dynamics of transcription factor populations. We find the pluripotent state of the network to be a steady state which is robust to global variations of gene switching rates which we argue are a good proxy for epigenetic states of individual promoters. The temporal dynamics of exiting the pluripotent state, on the other hand, is significantly influenced by the rates of genetic switching which makes cells more responsive to changes in extracellular signals.

Predictive and mechanistic multivariate linear regression models for reaction development.

Multivariate Linear Regression (MLR) models utilizing computationally-derived and empirically-derived physical organic molecular descriptors are described in this review. Several reports demonstrating the effectiveness of this methodological approach towards reaction optimization and mechanistic interrogation are discussed. A detailed protocol to access quantitative and predictive MLR models is provided as a guide for model development and parameter analysis.

Preserve Cultured Cell Cytonemes through a Modified Electron Microscopy Fixation.

Immunocytochemistry of cultured cells is a common and effective technique for determining compositions and localizations of proteins within cellular structures. However, traditional cultured cell fixation and staining protocols are not effective in preserving cultured cell cytonemes, long specialized filopodia that are dedicated to morphogen transport. As a result, limited mechanistic interrogation has been performed to assess their regulation. We developed a fixation protocol for cultured cells that preserves cytonemes, which allows for immunofluorescent analysis of endogenous and over-expressed proteins localizing to the delicate cellular structures.

Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement

SummaryFor kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells. Using this method, we performed a selectivity profiling for clinically relevant kinase inhibitors against 178 full-length kinases, and a mechanistic interrogation of the potency offsets observed between cellular and biochemical analysis. For the multikinase inhibitor crizotinib, our approach accurately predicted cellular potency and revealed improved target selectivity compared with biochemical measurements. Due to cellular ATP, a number of putative crizotinib targets are unexpectedly disengaged in live cells at a clinically relevant drug dose.

A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates.

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is a novel genome-editing system that has been successfully established in Aspergillus fumigatus. However, the current state of the technology relies heavily on DNA-based expression cassettes for delivering Cas9 and the guide RNA (gRNA) to the cell. Therefore, the power of the technology is limited to strains that are engineered to express Cas9 and gRNA. To overcome such limitations, we developed a simple and universal CRISPR-Cas9 system for gene deletion that works across different genetic backgrounds of A.fumigatus. The system employs in vitro assembly of dual Cas9 ribonucleoproteins (RNPs) for targeted gene deletion. Additionally, our CRISPR-Cas9 system utilizes 35 to 50bp of flanking regions for mediating homologous recombination at Cas9 double-strand breaks (DSBs). As a proof of concept, we first tested our system in the ΔakuB (ΔakuBku80 ) laboratory strain and generated high rates (97%) of gene deletion using 2µg of the repair template flanked by homology regions as short as 35bp. Next, we inspected the portability of our system across other genetic backgrounds of A.fumigatus, namely, the wild-type strain Af293 and a clinical isolate, A.fumigatus DI15-102. In the Af293 strain, 2µg of the repair template flanked by 35 and 50bp of homology resulted in highly efficient gene deletion (46% and 74%, respectively) in comparison to classical gene replacement systems. Similar deletion efficiencies were also obtained in the clinical isolate DI15-102. Taken together, our data show that in vitro-assembled Cas9 RNPs coupled with microhomology repair templates are an efficient and universal system for gene manipulation in A.fumigatus. IMPORTANCE Tackling the multifactorial nature of virulence and antifungal drug resistance in A.fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. Our simple and universal CRISPR-Cas9 system for gene manipulation generates efficient gene targeting across different genetic backgrounds of A.fumigatus. We anticipate that our system will simplify genome editing in A.fumigatus, allowing for the generation of single- and multigene knockout libraries. In addition, our system will facilitate the delineation of virulence factors and antifungal drug resistance genes in different genetic backgrounds of A.fumigatus.

Abstract 5795: Functional and mechanistic interrogation of BET Bromodomain degraders for the treatment of metastatic castration resistant prostate cancer

Abstract Metastatic castration-resistant prostate cancer (mCRPC), a lethal disease primarily driven by androgen receptor (AR) signaling, results in approximately 30,000 annual deaths in the U.S. While the AR is necessary for the function, survival, and differentiation of normal prostatic tissue, AR function and signaling shift from tumor suppressive to tumor promoting during prostate carcinogenesis. Surgical or chemical castration targeting the AR signaling axis has been the mainstay of prostate cancer (PCa) treatment. Unfortunately, despite castration, PCa will inevitably progress into castration resistant prostate cancer (CRPC) in which AR remains an important oncogene. Current CRPC therapeutics, such as second-generation AR antagonists, elicit only temporary responses and patients eventually succumb to the disease. Therefore, new therapeutics against the AR axis in CRPC are urgently needed. One such novel strategy for targeting the AR-pathway and inhibiting the growth of CRPC has been the use of bromodomain and extraterminal (BET) protein inhibitors such a JQ1, which disrupt AR and oncogenic c-MYC transcriptional activity. While BET inhibitors may seem to be attractive candidate drugs for clinical translation, their off-target effects, such as binding to other proteins, toxicity, and the rapid development of treatment resistance, limit their translation. However, a new class of molecules that target BET proteins through proteasomal degradation can improve efficacy and specificity over standard inhibitors. Based on our preliminary findings, we have hypothesized that pharmacologic BET degradation represents an important advance in CRPC treatment and may provide a novel therapeutic strategy that can enhance efficacy and disrupt resistance to AR-targeted therapy. Utilizing prostate cancer cell lines, organoids, and patient derived xenografts, we performed RNA-seq, ChIP-seq, and proteomic studies to assay the effects of BET inhibitors and degrader compounds in preclinical models. These inhibitors affected AR-positive prostate cancer cells preferentially over AR-negative cells, and proteomic and genomic mechanistic studies confirmed the disruption of oncogenic AR and MYC signaling both in vivo and in vitro. In conclusion, we are developing highly potent, small molecules that lead to the proteasomal-degradation of BET proteins. With optimized in vivo properties, BET degraders promise to be a novel potential therapeutic strategy for patients with mCRPC. Citation Format: Steven Kregel, Rohit Malik, Irfan A. Asangani, Ester Fernandez-Salas, Kari Wilder-Romans, Xia Jiang, Thekkelnaycke Rajendiran, Xuhong Cao, Corey Speers, Shaomeng Wang, Arul M. Chinnaiyan. Functional and mechanistic interrogation of BET Bromodomain degraders for the treatment of metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5795.

Abstract 3065: High-throughput screen identifies HSP90 as a potential therapeutic target in models of advanced CRPC

Abstract Castrate resistant prostate cancer (CRPC) remains the primary cause of mortality for prostate cancer (PCa) patients. Several therapies are currently approved for use against CRPC, consisting mostly of androgen receptor (AR) signaling inhibitors and toxic anti-microtubule chemotherapeutics that do not score long-term durable responses. Recent deep-sequencing analysis studies have elucidated the complex and heterogeneous landscape of CRPC, highlighting the necessity to develop effective targeted therapies against specific CRPC subtypes or therapies effective against numerous subtypes of CRPC. PTEN and p53 are two of the most frequently altered genes in CRPC, and are associated with therapy resistance and a poor clinical prognosis. The objective of this study was to identify and subsequently validate therapeutic targets against models of advanced PCa, specifically in the absence of PTEN and p53. Using non-biased high throughput screening technology, we identified HSP90 inhibitors to be potent and efficacious against a model of PTEN/p53 null PCa. HSP90 is a critical regulator of prostate cancer cell signaling homeostasis, and recently developed second generation compounds targeting HSP90 are effective against genotypically heterogeneous panels of cancer cell lines and have favorable safety parameters in the clinic. Screening results and subsequent validation efforts in vitro identified ganetespib as the most potent HSP90 inhibitor, with efficacy that translated into established human PCa cells lines. Ganetespib also displayed strongly inhibitory activity against PTEN/p53 null progenitor cells, a plastic subpopulation of PCa believed to be partly responsible for therapy resistance. In vivo, ganetespib completely inhibited progression of PIN to invasive adenocarcinoma in the Pten/Tp53 null PCa mouse model, leading to a significant reduction in tumor weight. Expanding from the mouse model to clinically predictive PDX-derived LuCaP models of human PCa, we found that ganetespib displayed a range of activity against a genotypically diverse array of 11 LuCaP organoids ex vivo. Furthermore, the PTEN null/p53 altered LuCaP 136 displayed a robust response to ganetespib in vivo, with ganetespib causing a significant reduction in tumor growth. Mechanistic interrogation in vitro, ex vivo, and in vivo revealed ganetespib induced effects to be multifactorial and model specific, with unifying trends being inactivation of PI3K signaling and modulation of cell cycle regulatory proteins. In all, these data indicate that inhibition of HSP90 is a novel therapeutic to treat advanced PCa via multifactorial perturbation of growth regulatory pathways. Citation Format: Keith H. Jansson, John Tucker, Lauren Stahl, Caitlyn Fuller, Michael Beshiri, John K. Simmons, Supreet Agarwal, Lei Fang, Aian Neil Alilin, Ross Lake, Crystal Tran, Paul G. Hynes, Jacob Cawley, JuanJuan Yin, Xiaohu Zhang, Rajarshi Guha, Shelley Hoover, R. Mark Simpson, Holly Nguyen, Eva Corey, Craig Thomas, David Proia, Kathleen Kelly. High-throughput screen identifies HSP90 as a potential therapeutic target in models of advanced CRPC [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 3065. doi:10.1158/1538-7445.AM2017-3065

Abstract 1254: Mechanistic interrogation of pre-treatment low dose aspirin effects in HER 2 positive breast cancer

Abstract Background: Prior data (Barron et al. Cancer Res. 2014 74:4065-77) suggests that pre-diagnostic exposure to aspirin can have significant effects on breast tumor biology and patient outcome. It has been proposed that aspirin inhibition of COX-2 may suppress lymphangiogenesis and metastasis (Karnezis et al Cancer Cell. 2014. 21:181-95). Here, we sought to recapitulate pre-diagnostic aspirin exposure in rodent models of Her2+ breast cancer and elucidate mechanisms of action. We also determined the effect of aspirin on tumor stroma, using a co-culture system of human tumor and mesenchymal stem cells (MSC). Methods: NOD/SCID mice were orthotopically implanted with Her2+ MDA-MB-231 or HCC1954 cells. 48hr later, animals began a daily low dose [30mg/kg or 120mg/kg] of aspirin, until tumors reached 250mm3. They were then resected. 3 weeks later, HCC1954 implanted animals were treated with trastuzumab (15mg/kg) and paclitaxel (5mg/kg) for 6 weeks. Primary tissues were analysed by immunohistochemistry to assess VEGF-C, -D, COX-2, LYVE1 and CD31. RNAseq was performed on tumours to identify aspirin perturbed molecular pathways. To determine the stromal response to aspirin, patient derived MSCs were cultured either alone or with HCC1954 cells and exposed to aspirin (2.5 or 7.5mM). Secreted VEGF-C was quantified. A tubule formation assay was performed to determine the impact of aspirin on angiogenesis. Pro-angiogenic protein expression was investigated using a human angiogenesis array platform. Results: A significant delay in tumor growth was observed in both tumor models following aspirin treatment (p<.01). Assessment of metastatic progression revealed that 120 mg/kg aspirin significantly (p<.05) increased time to metastasis and reduced primary regrowth in the MDA MB 231 model (p<.01). Immunohistochemical analysis of VEGF C, D and LYVE1 showed a significant dose dependant reduction (p<.01) in both models. RNAseq pathway analysis revealed a significant over-representation of mitochondrial electron transport chain genes. Downstream factors of AMPK showed significant (p<.01) upregulation suggesting alterations in metabolism. Aspirin (7.5mM) exposure resulted in loss of VEGF-C secretion from co-cultured tumor / MSC cell populations. Conditioned media harvested following aspirin treatment limited support tubule formation. Expression of pro-angiogenic factors in HCC1954 cells showed alterations following treatment, with the greatest decrease seen in Urokinase Plasminogen Activator (-42%) and its inhibitor Serpine1 (+55%). Conclusion: We have successfully recapitulated pre-treatment aspirin response in surgical resection models of Her2+ breast cancer, with IHC analysis confirming the impact of treatment on angiogenic and lymphangiogenic factors. RNAseq analysis implicates aspirin mediated alterations in cellular metabolism. Our data further reveals increased response to aspirin in stromal cell populations. Citation Format: Ian S. Miller, Sonja Khan, Liam P. Shiels, Sudipto Das, Bruce Moran, Finbarr P. Leacy, Paul M. Loadman, Robert S. Kerbel, Darran O' Connor, Kathleen Bennett, Róisín M. Dwyer, Annette T. Byrne. Mechanistic interrogation of pre-treatment low dose aspirin effects in HER 2 positive 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 1254. doi:10.1158/1538-7445.AM2017-1254