Patient derived tumor organoid (PDTO) models retain the structural, morphological, genetic, and clonal heterogeneity of the original tumors. The ability to efficiently generate, expand, and biobank PDTOs has the potential to make the clinical diversity of cancer accessible for personalized medicine assay guided therapeutic drug selection and drug discovery. We describe the miniaturization and growth in 96- and 384-well formats of a single non-tumor liver and two Hepatocellular carcinoma (HCC) organoids derived from cryopreserved PDTO cells and the application of high content imaging (HCI) to characterize the models and enhance drug sensitivity testing. Non-invasive sequentially acquired transmitted light images showed that seeding cryopreserved cells from non-tumoral and HCC PDTOs into 96- or 384-well plates in reduced growth factor Matrigel (rgf-MG) that were fed with growth medium every 3 days supported organoid growth up to 15 days. The number and sizes of organoids increased with longer times in culture. HCC PDTO's had more heterogeneous morphologies than non-tumor organoids with respect to size, shape, and optical density. Organoids cultured in rgf-MG could be stained in situ with HCI reagents without mechanical, chemical or enzymatic disruption of the hydrogel matrices and quantitative data extracted by image analysis. Hoechst and live/dead reagents provided organoid numbers and viability comparisons. HCC PDTO's stained with phalloidin or immuno-stained with α-tubulin antibodies revealed F-actin and microtubule cytoskeleton organization. HCC PDTO's stained with antibodies to signaling pathway proteins and their phosphorylation status allowed comparisons of relative expression levels and inference of pathway activation. Images of HCC PDTO's exposed to ellipticine showed that drugs penetrate Matrigel hydrogels and accumulate in organoid cells. 9-day 384-well HCC organoid cultures exhibited robust and reproducible growth signals suitable for cancer drug testing. Complimenting cell viability readouts with multiple HCI parameters including morphological features and dead cell staining improved the analysis of drug impact and enhanced the value that could be extracted from these more physiologically relevant three-dimensional HCC organoid cultures.

Development, optimization, and calibration of human transient receptor potential (TRP) channel Ca2+ mobilization assays for TRPM8, TRPV1, and TRPA1 are described. Heterologous expression of hTRPM8 in HEK293T cells was required for anti-TRPM8 antibody staining and TRPM8 agonist induced Ca2+ mobilization signals which were both used to optimize transfection efficiency. FLIPR Calcium 6 dye concentration, loading time, and TRPM8 transfected cell seeding density were optimized and a DMSO tolerance of ≤0.2% was set. The resting baseline relative fluorescent unit (RFUs) signals of the TRPM8 Ca2+ mobilization assay exhibited substantial well-to-well variability, even though such differences were small relative to maximal agonist induced responses. Maximum RFU, cumulative RFU sum, or area under the curve values were extracted from Ca2+ mobilization kinetic data to plot curves and calculate EC50 and IC50 values. Fold over baseline (FOB) ratio data processing eliminated well-to-well differences in resting baseline signals, reduced error bars, improved curve fits and reduced 95% confidence interval EC50 and IC50 ranges. FOB ratio data processing decreased variability and improved the precision of repeat measurements in single experimental sessions thereby reducing the minimum threshold difference in EC50 or IC50 values required to distinguish compound potencies. EC50 and IC50 values of TRPM8 agonists and antagonists determined in single experiments were strongly aligned to those from multiple independent experiments. Benchmark TRPM8, TRPV1, and TRPA1 EC50 and IC50 values were within the ranges previously reported for agonist and antagonist standards. The improved precision and accuracy of the TRP Ca2+ mobilization assays afforded by FOB ratio data processing enhances their utility for investigating structure activity relationships.

PCI-34051 is a valuable tool to interrogate the therapeutic effects of selective inhibition of HDAC8. However, it has not advanced to clinical trials, perhaps due to poor PK or off-target effects. We hypothesized that the presence of a hydroxamic acid (HA) group in PCI-34051 contributed to its lack of advancement. Therefore, we replaced the HA in the PCI-34051 scaffold with a series of moieties that have the potential to bind to Zn and evaluated their activity in a HDAC8 assay. Surprisingly, none of the replacements effectively mimicked the HA, and analogs lost significant potency. Evaluation of the analogs’ affinity to Zn indicated that none had affinity for Zn within the same range as the HA. These studies point to the difficulty in the application of bioisosteric replacements for Zn binding motifs.

Background and Purpose We extend the characterization of the TRPM8 antagonist VBJ103 with tests of selectivity, specificity and distribution, therapeutic efficacy of systemic administration against oxaliplatin-induced cold hyperalgesia and the impact of systemic administration on core body temperature (CBT). Experimental Approach Selectivity at human TRPA1 and TRPV1 as well as in vitro safety profiling was determined. Effects of systemic administration of VBJ103 were evaluated in a model of oxaliplatin-induced cold hyperalgesia. Both peripheral and centrally mediated effects of VBJ103 on CBT were assessed with radiotelemetry. Key Results VBJ103 had no antagonist activity at TRPV1 and TRPA1, but low potency TRPA1 activation. The only safety liability detected was partial inhibition of the dopamine transporter (DAT). VBJ103 delivered subcutaneously dose-dependently attenuated cold hypersensitivity in oxaliplatin-treated mice at 3, 10 and 30 mgkg(-1) (n = 7, P < 0.05). VBJ103 (30 mgkg(-1)) antinociception was influenced by neither the TRPA1 antagonist HC-030031 nor the DAT antagonist GBR12909. Subcutaneous administration of VBJ103 (3, 10 and 30 mgkg(-1), but not 100 or 300 mgkg(-1), n = 7) decreased CBT (2 degrees C). Intraperitoneal (i.p.) administration of VBJ103 (3, 10 and 30 mgkg-1) dose-dependently decreased CBT to an extent larger than that detected with subcutaneous administration. Intracerebroventricular (i.c.v.) administration (306 nmol/1 mu L; n = 5) did not alter CBT. Conclusions and Implications We achieve therapeutic efficacy with subcutaneous administration of a novel TRPM8 antagonist that attenuates deleterious influences on CBT, a side effect that has largely prevented the translation of TRPM8 as a target.

Three series of compounds were prioritized from a high content screening campaign that identified molecules that blocked dihydrotestosterone (DHT) induced formation of Androgen Receptor (AR) protein-protein interactions (PPIs) with the Transcriptional Intermediary Factor 2 (TIF2) coactivator and also disrupted preformed AR-TIF2 PPI complexes; the hydrobenzo-oxazepins (S1), thiadiazol-5-piperidine-carboxamides (S2), and phenyl-methyl-indoles (S3). Compounds from these series inhibited AR PPIs with TIF2 and SRC-1, another p160 coactivator, in mammalian 2-hybrid assays and blocked transcriptional activation in reporter assays driven by full length AR or AR-V7 splice variants. Compounds inhibited the growth of five prostate cancer cell lines, with many exhibiting differential cytotoxicity towards AR positive cell lines. Representative compounds from the 3 series substantially reduced both endogenous and DHT-enhanced expression and secretion of the prostate specific antigen (PSA) cancer biomarker in the C4-2 castration resistant prostate cancer (CRPC) cell line. The comparatively weak activities of series compounds in the H3-DHT and/or TIF2 box 3 LXXLL-peptide binding assays to the recombinant ligand binding domain of AR suggest that direct antagonism at the orthosteric ligand binding site or AF-2 surface respectively are unlikely mechanisms of action. Cellular enhanced thermal stability assays (CETSA) indicated that compounds engaged AR and reduced the maximum efficacy and right shifted the EC50 of DHT-enhanced AR thermal stabilization consistent with the effects of negative allosteric modulators. Molecular docking of potent representative hits from each series to AR structures suggest that S1-1 and S2-6 engage a novel binding pocket (BP-1) adjacent to the orthosteric ligand binding site, while S3-11 occupies the AR binding function 3 (BF-3) allosteric pocket. Hit binding poses indicate spaces and residues adjacent to the BP-1 and BF-3 pockets that will be exploited in future medicinal chemistry optimization studies. Small molecule allosteric modulators that prevent/disrupt AR PPIs with coactivators like TIF2 to alter transcriptional activation in the presence of orthosteric agonists might evade the resistance mechanisms to existing prostate cancer drugs and provide novel starting points for medicinal chemistry lead optimization and future development into therapies for metastatic CRPC.

Acute kidney injury (AKI), a sudden loss of kidney function, is a common and serious condition for which there are no approved specific therapies. While there are multiple approaches to treat the underlying causes of AKI, no targets have been clinically validated. Here, we assessed a series of potent, selective competitive inhibitors of histone deacetylase 8 (HDAC8), a promising therapeutic target in an AKI setting. Using biochemical assays, zebrafish AKI phenotypic assays, and human kidney organoid assays, we show that selective HDAC8 inhibitors can lead to efficacy in increasingly stringent models. One of these, PCI-34051, was efficacious in a rodent model of AKI, further supporting the potential for HDAC8 inhibitors and, in particular, this scaffold as a therapeutic approach to AKI.

In solid tumors like head and neck cancer (HNC), chronic and acute hypoxia have serious adverse clinical consequences including poorer overall patient prognosis, enhanced metastasis, increased genomic instability, and resistance to radiation-, chemo-, or immuno-therapies. However, cells in the two-dimensional monolayer cultures typically used for cancer drug discovery experience 20%-21% O2 levels (normoxic) which are 4-fold higher than O2 levels in normal tissues and ≥10-fold higher than in the hypoxic regions of solid tumors. The oxygen electrodes, exogenous bio-reductive markers, and increased expression of endogenous hypoxia-regulated proteins like HIF-1α generally used to mark hypoxic regions in solid tumors are impractical in large sample numbers and longitudinal studies. We used a novel homogeneous live-cell permeant HypoxiTRAK™ (HPTK) molecular probe compatible with high content imaging detection, analysis, and throughput to identify and quantify hypoxia levels in live HNC multicellular tumor spheroid (MCTS) cultures over time. Accumulation of fluorescence HPTK metabolite in live normoxic HNC MCTS cultures correlated with hypoxia detection by both pimonidazole and HIF-1α staining. In HNC MCTSs, hypoxic cytotoxicity ratios for the hypoxia activated prodrugs (HAP) evofosfamide and tirapazamine were much smaller than have been reported for uniformly hypoxic 2D monolayers in gas chambers, and many viable cells remained after HAP exposure. Cells in solid tumors and MCTSs experience three distinct O2 microenvironments dictated by their distances from blood vessels or MCTS surfaces, respectively; oxic, hypoxic, or intermediate levels of hypoxia. These studies support the application of more physiologically relevant in vitro 3D models that recapitulate the heterogeneous microenvironments of solid tumors for preclinical cancer drug discovery.

We describe the development, optimization, and validation of 384-well growth inhibition assays for six patient-derived melanoma cell lines (PDMCLs), three wild type (WT) for BRAF and three with V600E-BRAF mutations. We conducted a pilot drug combination (DC) high-throughput screening (HTS) of 45 pairwise 4×4 DC matrices prepared from 10 drugs in the PDMCL assays: two B-Raf inhibitors (BRAFi), a MEK inhibitor (MEKi), and a methylation agent approved for melanoma; cytotoxic topoisomerase II and DNA methyltransferase chemotherapies; and drugs targeting the base excision DNA repair enzyme APE1 (apurinic/apyrimidinic endonuclease-1/redox effector factor-1), SRC family tyrosine kinases, the heat shock protein 90 (HSP90) molecular chaperone, and histone deacetylases.Pairwise DCs between dasatinib and three drugs approved for melanoma therapy—dabrafenib, vemurafenib, or trametinib—were flagged as synergistic in PDMCLs. Exposure to fixed DC ratios of the SRC inhibitor dasatinib with the BRAFis or MEKis interacted synergistically to increase PDMCL sensitivity to growth inhibition and enhance cytotoxicity independently of PDMCL BRAF status. These DCs synergistically inhibited the growth of mouse melanoma cell lines that either were dabrafenib-sensitive or had acquired resistance to dabrafenib with cross resistance to vemurafenib, trametinib, and dasatinib. Dasatinib DCs with dabrafenib, vemurafenib, or trametinib activated apoptosis and increased cell death in melanoma cells independently of their BRAF status or their drug resistance phenotypes. These preclinical in vitro studies provide a data-driven rationale for the further investigation of DCs between dasatinib and BRAFis or MEKis as candidates for melanoma combination therapies with the potential to improve outcomes and/or prevent or delay the emergence of disease resistance.

Screening certain environmental chemicals for their ability to interact with endocrine targets, including the androgen receptor (AR), is an important global concern. We previously developed a model using a battery of eleven in vitro AR assays to predict in vivo AR activity. Here we describe a revised mathematical modeling approach that also incorporates data from newly available assays and demonstrate that subsets of assays can provide close to the same level of predictivity. These subset models are evaluated against the full model using 1820 chemicals, as well as in vitro and in vivo reference chemicals from the literature. Agonist batteries of as few as six assays and antagonist batteries of as few as five assays can yield balanced accuracies of 95% or better relative to the full model. Balanced accuracy for predicting reference chemicals is 100%. An approach is outlined for researchers to develop their own subset batteries to accurately detect AR activity using assays that map to the pathway of key molecular and cellular events involved in chemical-mediated AR activation and transcriptional activity. This work indicates in vitro bioactivity and in silico predictions that map to the AR pathway could be used in an integrated approach to testing and assessment for identifying chemicals that interact directly with the mammalian AR.

Abstract Current in vitro models of primary liver cancer insufficiently recapitulates the tumor heterogeneity and pathophysiology of the original patient's tumor. This significantly hinders translational studies and often leads to the high rate of clinical trial attrition and poor patient response. Recent technological advances in organoid culture methods have been reported to overcome these limitations. Our group has successfully established 3 patient-derived HCC tumor organoids that closely recapitulate the patient's pathophysiology. Conventional short-term monolayer culture of the same 3 tumors as well as 3 non-tumoral adjacent liver and 3 normal liver organoids were established and compared with 5 HCC cell lines from ATCC for their drug response profiles. Subsequent drug screening was performed using a panel of 100 drugs, of which 70 are FDA-approved for either HCC or other cancers, as well as 30 pipeline compounds targeting frequently mutated pathways in HCC. We found that our screening pipeline could achieve robust z' scores and reproducible data points with technical and biological replicates. Principal component analysis of Total Area under curve (AUC) values show distinct and progressive changes between Normal Liver, Non-tumoral adjacent liver, and Tumor organoids. Upon comparison with HCC cell lines Hep3B, HepG2, Huh7, SNU398 and PLC5, we found HCC patient-derived short term cultures, particularly organoids have more diverse drug sensitivity profiles and may be a better indicator of patient heterogeneity. Interestingly, FDA-approved drugs for HCC such as Regorafenib show high sensitivity and tumor specificity to ATCC cell lines (ATCC vs Normal liver P=0.00017***), but this specificity was reduced and lost in patient-derived 2D cultures (P=0.045*) and organoids (P=0.895), respectively. This may explain the observed hepatotoxicity observed in HCC patients and warrant further investigation of more targeted ways of treating HCC. Citation Format: Alissa Michelle Go Wong, Winnie Cheung, Maggie Wong, Aikha Wong, Helena Lam, Zhe Zhang, Matthew Man, Hui Chen, David Close, Paul Johnston, Anthony Chan, Paul Lai, Joseph Kwong, Nathalie Wong. High-throughput screening of patient-derived HCC organoids for drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3911.

With approval rates <5% and the probability of success in oncology clinical trials of 3.4%, more physiologically relevant in vitro three-dimensional models are being deployed during lead generation to select better drug candidates for solid tumors. Multicellular tumor spheroids (MCTSs) resemble avascular tumor nodules, micrometastases, or the intervascular regions of large solid tumors with respect to morphology, cell-cell and cell-extracellular matrix contacts, and volume growth kinetics. MCTSs develop gradients of nutrient and oxygen concentration resulting in diverse microenvironments with differential proliferation and drug distribution zones. We produced head and neck squamous cell carcinoma (HNSCC) MCTSs in 384-well U-bottom ultra-low-attachment microtiter plates and used metabolic viability and imaging methods to measure morphologies, growth phenotypes and the effects of 19 anticancer drugs. We showed that cell viability measurements underestimated the impact of drug exposure in HNSCC MCTS cultures, but that incorporating morphology and dead-cell staining analyses increased the number of drugs judged to have substantially impacted MCTS cultures. A cumulative multiparameter drug impact score enabled us to stratify MCTS drug responses into high-, intermediate-, and low-impact tiers, and maximized the value of these more physiologically relevant tumor cultures. It is conceivable that the viable cells present in MCTS cultures after drug exposure arise from drug-resistant populations that could represent a source of drug failure and recurrence. Long-term monitoring of treated MCTS cultures could provide a strategy to determine whether these drug-resistant populations represent circumstances where tumor growth is delayed and may ultimately give rise to regrowth.

Animal and clinical studies demonstrate that cancer drug combinations (DCs) are more effective than single agents. However, it is difficult to predict which DCs will be more efficacious than individual drugs. Systematic DC high-throughput screening (HTS) of 100 approved drugs in the National Cancer Institute’s panel of 60 cancer cell lines (NCI-60) produced data to help select DCs for further consideration. We miniaturized growth inhibition assays into 384-well format, increased the fetal bovine serum amount to 10%, lengthened compound exposure to 72 h, and used a homogeneous detection reagent. We determined the growth inhibition 50% values of individual drugs across 60 cell lines, selected drug concentrations for 4 × 4 DC matrices (DCMs), created DCM master and replica daughter plate sets, implemented the HTS, quality control reviewed the data, and analyzed the results. A total of 2620 DCMs were screened in 60 cancer cell lines to generate 3.04 million data points for the NCI ALMANAC (A Large Matrix of Anti-Neoplastic Agent Combinations) database. We confirmed in vitro a synergistic drug interaction flagged in the DC HTS between the vinca-alkaloid microtubule assembly inhibitor vinorelbine (Navelbine) tartrate and the epidermal growth factor-receptor tyrosine kinase inhibitor gefitinib (Iressa) in the SK-MEL-5 melanoma cell line. Seventy-five percent of the DCs examined in the screen are not currently in the clinical trials database. Selected synergistic drug interactions flagged in the DC HTS described herein were subsequently confirmed by the NCI in vitro, evaluated mechanistically, and were shown to have greater than single-agent efficacy in mouse xenograft human cancer models. Enrollment is open for two clinical trials for DCs that were identified in the DC HTS. The NCI ALMANAC database therefore constitutes a valuable resource for selecting promising DCs for confirmation, mechanistic studies, and clinical translation.

The eight mammalian Src-family tyrosine kinases are dynamic, multi-domain structures, which adopt distinct "open" and "closed" conformations. In the closed conformation, the regulatory SH3 and SH2 domains pack against the back of the kinase domain, providing allosteric control of kinase activity. Small molecule ligands that engage the regulatory SH3-SH2 region have the potential to modulate Src-family kinase activity for therapeutic advantage. Here we describe an HTS-compatible fluorescence polarization assay to identify small molecules that interact with the unique-SH3-SH2-linker (U32L) region of Hck, a Src-family member expressed exclusively in cells of myeloid lineage. Hck has significant potential as a drug target in acute myeloid leukemia, an aggressive form of cancer with substantial unmet clinical need. The assay combines recombinant Hck U32L protein with a fluorescent probe peptide that binds to the SH3 domain in U32L, resulting in an increased FP signal. Library compounds that interact with the U32L protein and interfere with probe binding reduce the FP signal, scoring as hits. Automated 384-well high-throughput screening of 60,000 compounds yielded Z'-factor coefficients > 0.7 across nearly 200 assay plates, and identified a series of hit compounds with a shared pyrimidine diamine substructure. Surface plasmon resonance assays confirmed direct binding of hit compounds to the Hck U32L target protein as well as near-full-length Hck. Binding was not observed with the individual SH3 and SH2 domains, demonstrating that these compounds recognize a specific three-dimensional conformation of the regulatory regions. This conclusion is supported by computational docking studies, which predict ligand contacts with a pocket formed by the juxtaposition of the SH3 domain, the SH3-SH2 domain connector, and the SH2-kinase linker. Each of the four validated hits stimulated recombinant, near-full-length Hck activity in vitro, providing evidence for allosteric effects on the kinase domain. These results provide a path to discovery and development of chemical scaffolds to target the regulatory regions of Hck and other Src family kinases as a new approach to pharmacological kinase control.

Systematic unbiased high-throughput screening (HTS) of drug combinations (DCs) in well-characterized tumor cell lines is a data-driven strategy to identify novel DCs with potential to be developed into effective therapies. Four DCs from a DC HTS campaign were selected for confirmation; only one appears in clinicaltrials.gov and limited preclinical in vitro data indicates that the drug pairs interact synergistically. Nineteen DC-tumor cell line sets were confirmed to interact synergistically in three pharmacological interaction models. We developed an imaging assay to quantify accumulation of the ABCG2 efflux transporter substrate Hoechst. Gefitinib and raloxifene enhanced Hoechst accumulation in ABCG2 (BCRP)-expressing cells, consistent with inhibition of ABCG2 efflux. Both drugs also inhibit ABCB1 efflux. Mitoxantrone, daunorubicin, and vinorelbine are substrates of one or more of the ABCG2, ABCB1, or ABCC1 efflux transporters expressed to varying extents in the selected cell lines. Interactions between ABC drug efflux transporter inhibitors and substrates may have contributed to the observed synergy; however, other mechanisms may be involved. Novel synergistic DCs identified by HTS were confirmed in vitro, and plausible mechanisms of action studied. Similar approaches may justify the testing of novel HTS-derived DCs in mouse xenograft human cancer models and support the clinical evaluation of effective in vivo DCs in patients.

The poor success rate of cancer drug discovery has prompted efforts to develop more physiologically relevant cellular models for early preclinical cancer lead discovery assays. For solid tumors, this would dictate the implementation of three-dimensional (3D) tumor models that more accurately recapitulate human solid tumor architecture and biology. A number of anchorage-dependent and anchorage-independent in vitro 3D cancer models have been developed together with homogeneous assay methods and high content imaging approaches to assess tumor spheroid morphology, growth, and viability. However, several significant technical challenges have restricted the implementation of some 3D models in HTS. We describe a method that uses 384-well U-bottomed ultra-low attachment (ULA) microplates to produce head and neck tumor spheroids for cancer drug discovery assays. The production of multicellular head and neck cancer spheroids in 384-well ULA-plates occurs in situ, does not impose an inordinate tissue culture burden for HTS, is readily compatible with automation and homogeneous assay detection methods, and produces high-quality uniform-sized spheroids that can be utilized in cancer drug cytotoxicity assays within days rather than weeks.

Twenty percent of prostate cancer (PCa) patients develop a noncurable drug-resistant form of the disease termed castration-resistant prostate cancer (CRPC). Overexpression of Androgen Receptor (AR) coactivators such as transcriptional intermediary factor 2 (TIF2) is associated with poor CRPC patient outcomes. We describe the implementation of the AR-TIF2 protein-protein interaction biosensor (PPIB) assay in a high-content screening (HCS) campaign of 143,535 compounds. The assay performed robustly and reproducibly and enabled us to identify compounds that inhibited dihydrotestosterone (DHT)-induced AR-TIF2 protein-protein interaction (PPI) formation or disrupted preexisting AR-TIF2 PPIs. We used multiparameter HCS data z-scores to identify and deprioritize cytotoxic or autofluorescent outliers and confirmed the resulting qualified actives in triplicate. None of the confirmed AR-TIF2 PPIB inhibitors/disruptors exhibited activity in a p53-hDM2 PPIB counter screen, indicating that they were unlikely to be either nonselective PPI inhibitors or to interfere with the biosensor assay format. However, eight confirmed AR-TIF2 PPIB actives also inhibited the glucocorticoid receptor (GR) nuclear translocation counter screen by >50%. These compounds were deprioritized because they either lacked AR specificity/selectivity, or they inhibited a shared component of the AR and GR signaling pathways. Twenty-nine confirmed AR-TIF2 PPIB actives also inhibited the AR nuclear localization counter screen, suggesting that they might indirectly inhibit the AR-TIF2 PPIB assay rather than directly blocking/disrupting PPIs. A total of 62.2% of the confirmed actives inhibited the DHT-induced AR-TIF2 PPI formation in a concentration-dependent manner with IC(50)s<40 mu M, and 59.4% also disrupted preexisting AR-TIF2 PPI complexes. Overall, the hit rate for the AR-TIF2 PPIB HCS campaign was 0.12%, and most hits inhibited AR-TIF2 PPI formation and disrupted preexisting AR-TIF2 complexes with similar AR-red fluorescent protein distribution phenotypes. Further secondary and tertiary hit characterization assays are underway to select AR-TIF2 PPI inhibitor/disruptor hits suitable for medicinal chemistry lead optimization and development into novel PCa/CRPC therapeutics.

Multicellular tumor spheroid (MCTS) cultures represent more physiologically relevant in vitro cell tumor models that recapitulate the microenvironments and cell-cell or cell-extracellular matrix interactions which occur in solid tumors. We characterized the morphologies, viability, and growth behaviors of MCTSs produced by 11 different head and neck squamous cell carcinoma (HNSCC) cell lines seeded into and cultured in ultra-low attachment microtiter plates (ULA-plates) over extended periods of time. HNSCC MCTS cultures developed microenvironments, which resulted in differences in proliferation rates, metabolic activity, and mitochondrial functional activity between cells located in the outer layers of the MCTS and cells in the interior. HNSCC MCTS cultures exhibited drug penetration and distribution gradients and some developed necrotic cores. Perhaps the most profound effect of culturing HNSCC cell lines in MCTS cultures was their dramatically altered and varied growth phenotypes. Instead of the exponential growth that are characteristic of two-dimensional HNSCC growth inhibition assays, some MCTS cultures displayed linear growth rates, categorized as rapid, moderate, or slow, dormant MCTSs remained viable but did not grow, and some MCTSs exhibited death phenotypes that were either progressive and slow or rapid. The ability of MCTS cultures to develop microenvironments and to display a variety of different growth phenotypes provides in vitro models that are more closely aligned with solid tumors in vivo. We anticipate that the implementation MCTS models to screen for new cancer drugs for solid tumors like HNSCC will produce leads that will translate better in in vivo animal models and patients.

Studies indicate that elevated interleukin-6 (IL-6) levels engage IL6Rα-gp130 receptor complexes to activate signal transducer and activator of transcription 3 (STAT3) that is hyperactivated in many cancers including head and neck squamous cell carcinoma (HNSCC). Our previous HCS campaign identified several hits that selectively blocked IL-6-induced STAT3 activation. This study describes our investigation of the mechanism(s) of action of three of the four chemical series that progressed to lead activities: a triazolothiadiazine (864669), amino alcohol (856350), and an oxazole-piperazine (4248543). We demonstrated that all three blocked IL-6-induced upregulation of the cyclin D1 and Bcl-XL STAT3 target genes. None of the compounds exhibited direct binding interactions with STAT3 in surface plasmon resonance (SPR) binding assays; neither did they inhibit the recruitment and binding of a phospho-tyrosine-gp130 peptide to STAT3 in a fluorescence polarization assay. Furthermore, they exhibited little or no inhibition in a panel of 83 cancer-associated in vitro kinase profiling assays, including lack of inhibition of IL-6-induced Janus kinase (JAK 1, 2, and 3) activation. Further, 864669 and 4248543 selectively inhibited IL-6-induced STAT3 activation but not that induced by oncostatin M (OSM). The compounds 864669 and 4248543 abrogated IL-6-induced phosphorylation of the gp130 signaling subunit (phospho-gp130Y905) of the IL-6-receptor complex in HNSCC cell lines which generate docking sites for the SH2 domains of STAT3. Our data indicate that 864669 and 4248543 block IL-6-induced STAT activation by interfering with the recruitment, assembly, or activation of the hexamer-activated IL-6/IL-6Rα/gp130 signaling complex that occurs after IL-6 binding to IL-6Rα subunits.

High cancer drug development attrition rates have provoked considerable debate about whether the two-dimensional tumor growth inhibition high-throughput screening assays used in pre-clinical lead discovery adequately reflect solid tumor complexity. We used automated high-content screening image acquisition and analysis methods to compare fluorescent drug uptake, accumulation, and distribution in Cal33 and FaDu head and neck cancer (HNC) monolayer and multicellular tumor spheroid (MCTS) models. Ellipticine, idarubicin, daunorubicin, and doxorubicin were studied because of their fluorescent properties and broad anti-tumor activities. HNC MCTSs were generated in 384-well ultra-low attachment plates where compound exposure, image acquisition, and analysis could be performed in situ. Fluorescent drug accumulation in Cal33 monolayer and MCTS cultures was linear with respect to concentration, and appeared to achieve steady-state levels within 10-15 min of drug exposure, which were maintained through 30-45 min. Drug accumulation in monolayers was independent of cell number and/or density, and every cell achieved uniform drug concentrations. In MCTSs, however, drug accumulation increased as the number of cells and sizes of the MCTSs became bigger. Drugs exhibited restricted penetration and distribution gradients, accumulating preferentially in cells in the outer layers of MCTSs relative to those in the inner cores. Cal33 monolayers were 6-, 20-, 10-, and 16-fold more sensitive than MCTSs to growth inhibition by ellipticine, idarubicin, daunorubicin, and doxorubicin, respectively. In Cal33 MCTSs exposed to ellipticine or doxorubicin for 24 h, MCTSs were smaller and although they still exhibited drug penetration and distribution gradients, the fluorescent intensity difference between outer and inner cells was reduced. After a 24 h exposure, both drugs had penetrated throughout FaDu MCTSs, consistent with drug-induced death of peripheral cell layers enhancing drug penetration. The increased resistance of MCTS cultures and their ability to recapitulate drug penetration and distribution gradients argues strongly for the deployment of these more physiological models in cancer lead discovery. MCTSs have the potential to enhance the correlation between in vitro potencies and in vivo efficacy, and ultimately may lead to improved cancer drug approval rates.

The continued activation of androgen receptor (AR) transcription and elevated expression of AR and transcriptional intermediary factor 2 (TIF2) coactivator observed in prostate cancer (CaP) recurrence and the development of castration-resistant CaP (CRPC) support a screening strategy for small-molecule inhibitors of AR-TIF2 protein-protein interactions (PPIs) to find new drug candidates. Small molecules can elicit tissue selective effects, because the cells of distinct tissues express different levels and cohorts of coregulatory proteins. We reconfigured the AR-TIF2 PPI biosensor (PPIB) assay in the PC-3 CaP cell line to determine whether AR modulators and hits from an AR-TIF2 PPIB screen conducted in U-2 OS cells would behave differently in the CaP cell background. Although we did not observe any significant differences in the compound responses between the assay performed in osteosarcoma and CaP cells, the U-2 OS AR-TIF2 PPIB assay would be more amenable to screening, because both the virus and cell culture demands are lower. We implemented a testing paradigm of counter-screens and secondary hit characterization assays that allowed us to identify and deprioritize hits that inhibited/disrupted AR-TIF2 PPIs and AR transcriptional activation (AR-TA) through antagonism of AR ligand binding or by non-specifically blocking nuclear receptor trafficking. Since AR-TIF2 PPI inhibitor/disruptor molecules act distally to AR ligand binding, they have the potential to modulate AR-TA in a cell-specific manner that is distinct from existing anti-androgen drugs, and to overcome the development of resistance to AR antagonism. We anticipate that the application of this testing paradigm to characterize the hits from an AR-TIF2 PPI high-content screening campaign will enable us to prioritize the AR-TIF2 PPI inhibitor/disruptor leads that have potential to be developed into novel therapeutics for CaP and CRPC.