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Supplementary Figure 1 from An EGFR–Src–Arg–Cortactin Pathway Mediates Functional Maturation of Invadopodia and Breast Cancer Cell Invasion

Supplementary Figures Legends 1-6 from An EGFR–Src–Arg–Cortactin Pathway Mediates Functional Maturation of Invadopodia and Breast Cancer Cell Invasion

Supplementary Figure 5 from An EGFR–Src–Arg–Cortactin Pathway Mediates Functional Maturation of Invadopodia and Breast Cancer Cell Invasion

Supplementary Figure 3 from An EGFR–Src–Arg–Cortactin Pathway Mediates Functional Maturation of Invadopodia and Breast Cancer Cell Invasion

Supplementary Figure 4 from An EGFR–Src–Arg–Cortactin Pathway Mediates Functional Maturation of Invadopodia and Breast Cancer Cell Invasion

Anti-cancer uses of non-oncology drugs have occasionally been found, but such discoveries have been serendipitous. We sought to create a public resource containing the growth inhibitory activity of 4,518 drugs tested across 578 human cancer cell lines. We used PRISM, a molecular barcoding method, to screen drugs against cell lines in pools. An unexpectedly large number of non-oncology drugs selectively inhibited subsets of cancer cell lines in a manner predictable from the cell lines' molecular features. Our findings include compounds that killed by inducing PDE3A-SLFN12 complex formation; vanadium-containing compounds whose killing depended on the sulfate transporter SLC26A2; the alcohol dependence drug disulfiram, which killed cells with low expression of metallothioneins; and the anti-inflammatory drug tepoxalin, which killed via the multi-drug resistance protein ABCB1. The PRISM drug repurposing resource (https://depmap.org/repurposing) is a starting point to develop new oncology therapeutics, and more rarely, for potential direct clinical translation.

Adherent cells cultured on flat, homogeneous surfaces typically maintain an intact cell body with a polygonal or fan shape, despite active migration and strong mechanical interactions with the substratum. We hypothesized that, in addition to the constraint of the surface membrane, an active mechanism may be involved in maintaining the shape and integrity of the cell body particularly where cells encounter complex topographic patterns of guidance cues. To detect if there is a mechanism that constrains cell shape, we plated NIH 3T3 fibroblasts on ring-patterned substrata 8–17.5 microns in width and 53–133 microns in outer diameter. Untreated cells have a limited angular span, encompassing an average of 108 degrees around the ring, even though these cells were able to cover a much larger surface when plated on flat surfaces of the same material. Treatment of 3T3 cells with a myosin II inhibitor, blebbistatin, induced a striking increase in the bending ability, causing cells to cover more than 60% of the ring. Inhibition of the Rho-dependent kinase with Y-27632 caused a similar but smaller increase in the angular span. Our results suggest that cell shape is controlled not only by the passive constraint of the surface membrane but also by an active mechanism driven by myosin II-mediated contractility under the regulation of Rho-dependent kinase. The inward surface tension-like forces allow the cell to maintain its integrity while navigating through complex physiological environments.

Standardized protocols for wastewater-based surveillance (WBS) for the RNA of SARS-CoV-2, the virus responsible for the current COVID-19 pandemic, are being developed and refined worldwide for early detection of disease outbreaks. We report here on lessons learned from establishing a WBS program for SARS-CoV-2 integrated with a human surveillance program for COVID-19. We have established WBS at three campuses of a university, including student residential dormitories and a hospital that treats COVID-19 patients. Lessons learned from this WBS program address the variability of water quality, new detection technologies, the range of detectable viral loads in wastewater, and the predictive value of integrating environmental and human surveillance data. Data from our WBS program indicated that water quality was statistically different between sewer sampling sites, with more variability observed in wastewater coming from individual buildings compared to clusters of buildings. A new detection technology was developed based upon the use of a novel polymerase called V2G. Detectable levels of SARS-CoV-2 in wastewater varied from 102 to 106 genomic copies (gc) per liter of raw wastewater (L). Integration of environmental and human surveillance data indicate that WBS detection of 100 gc/L of SARS-CoV-2 RNA in wastewater was associated with a positivity rate of 4% as detected by human surveillance in the wastewater catchment area, though confidence intervals were wide (β ~ 8.99 ∗ ln(100); 95% CI = 0.90–17.08; p < 0.05). Our data also suggest that early detection of COVID-19 surges based on correlations between viral load in wastewater and human disease incidence could benefit by increasing the wastewater sample collection frequency from weekly to daily. Coupling simpler and faster detection technology with more frequent sampling has the potential to improve the predictive potential of using WBS of SARS-CoV-2 for early detection of the onset of COVID-19., Graphical abstract Unlabelled Image

The Library of Integrated Network-based Cellular Signatures (LINCS) program is a national consortium funded by the NIH to generate a diverse and extensive reference library of cell-based perturbation-response signatures, along with novel data analytics tools to improve our understanding of human diseases at the systems level. In contrast to other large-scale data generation efforts, LINCS Data and Signature Generation Centers (DSGCs) employ a wide range of assay technologies cataloging diverse cellular responses. Integration of, and unified access to LINCS data has therefore been particularly challenging. The Big Data to Knowledge (BD2K) LINCS Data Coordination and Integration Center (DCIC) has developed data standards specifications, data processing pipelines, and a suite of end-user software tools to integrate and annotate LINCS-generated data, to make LINCS signatures searchable and usable for different types of users. Here, we describe the LINCS Data Portal (LDP) (http://lincsportal.ccs.miami.edu/), a unified web interface to access datasets generated by the LINCS DSGCs, and its underlying database, LINCS Data Registry (LDR). LINCS data served on the LDP contains extensive metadata and curated annotations. We highlight the features of the LDP user interface that is designed to enable search, browsing, exploration, download and analysis of LINCS data and related curated content.

Phosphorylation of cortactin downstream of the EGF receptor–Src-Arg kinase cascade triggers maturation of invadopodia, actin-rich protrusions that breast cancer cells use to invade the extracellular matrix. Phosphocortactin recruits Vav2 to invadopodia to activate Rac3 and support actin polymerization, matrix degradation, and invasion., Breast carcinoma cells use specialized, actin-rich protrusions called invadopodia to degrade and invade through the extracellular matrix. Phosphorylation of the actin nucleation–promoting factor and actin-stabilizing protein cortactin downstream of the epidermal growth factor receptor–Src-Arg kinase cascade is known to be a critical trigger for invadopodium maturation and subsequent cell invasion in breast cancer cells. The functions of cortactin phosphorylation in this process, however, are not completely understood. We identify the Rho-family guanine nucleotide exchange factor Vav2 in a comprehensive screen for human SH2 domains that bind selectively to phosphorylated cortactin. We demonstrate that the Vav2 SH2 domain binds selectively to phosphotyrosine-containing peptides corresponding to cortactin tyrosines Y421 and Y466 but not to Y482. Mutation of the Vav2 SH2 domain disrupts its recruitment to invadopodia, and an SH2-domain mutant form of Vav2 cannot support efficient matrix degradation in invasive MDA-MB-231 breast cancer cells. We show that Vav2 function is required for promoting invadopodium maturation and consequent actin polymerization, matrix degradation, and invasive migratory behavior. Using biochemical assays and a novel Rac3 biosensor, we show that Vav2 promotes Rac3 activation at invadopodia. Rac3 knockdown reduces matrix degradation by invadopodia, whereas a constitutively active Rac3 can rescue the deficits in invadopodium function in Vav2-knockdown cells. Together these data indicate that phosphorylated cortactin recruits Vav2 to activate Rac3 and promote invadopodial maturation in invasive breast cancer cells.

Cultured cell lines are the workhorse of cancer research, but it is unclear to what extent they recapitulate the cellular heterogeneity observed among malignant cells in tumors, given the absence of a native tumor microenvironment. Here, we used multiplexed single cell RNA-seq to profile ~200 cancer cell lines. We uncovered expression programs that are recurrently heterogeneous within many cancer cell lines and are largely independent of observed genetic diversity. These programs of heterogeneity are associated with diverse biological processes, including cell cycle, senescence, stress and interferon responses, epithelial-to-mesenchymal transition, and protein maturation and degradation. Notably, some of these recurrent programs recapitulate those seen in human tumors, suggesting a prominent role of intrinsic plasticity in generating intra-tumoral heterogeneity. Moreover, the data allowed us to prioritize specific cell lines as model systems of cellular plasticity. We used two such models to demonstrate the dynamics, regulation and drug sensitivities associated with a cancer senescence program also observed in human tumors. Our work describes the landscape of cellular heterogeneity in diverse cancer cell lines, and identifies recurrent patterns of expression heterogeneity that are shared between tumors and specific cell lines and can thus be further explored in follow up studies.

Anti-cancer uses of non-oncology drugs have been found on occasion, but such discoveries have been serendipitous and rare. We sought to create a public resource containing the growth inhibitory activity of 4,518 drugs tested across 578 human cancer cell lines. To accomplish this, we used PRISM, which involves drug treatment of molecularly barcoded cell lines in pools. Relative barcode abundance following treatment thus reflects cell line viability. We found that an unexpectedly large number of non-oncology drugs selectively inhibited subsets of cancer cell lines. Moreover, the killing activity of the majority of these drugs was predictable based on the molecular features of the cell lines. Follow-up of several of these compounds revealed novel mechanisms. For example, compounds that kill by inducing PDE3A-SLFN12 complex formation; vanadium-containing compounds whose killing is dependent on the sulfate transporter SLC26A2; the alcohol dependence drug disulfiram, which kills cells with low expression of metallothioneins; and the anti-inflammatory drug tepoxalin, whose killing is dependent on high expression of the multi-drug resistance gene ABCB1. These results illustrate the potential of the PRISM drug repurposing resource as a starting point for new oncology therapeutic development. The resource is available at https://depmap.org.

Natural killer (NK) cells exhibit potent activity in pre-clinical models of diverse hematologic malignancies and solid tumors and infusion of high numbers of NK cells, either autologous or allogeneic, after their ex vivo expansion and activation, has been feasible and safe in clinical studies. To systematically define molecular features in human tumor cells which determine their degree of sensitivity to human allogeneic NK cells, we quantified the NK cell responsiveness of hundreds of molecularly-annotated “DNA-barcoded” solid tumor cell lines in multiplexed format (PRISM; Profiling Relative Inhibition Simultaneously in Mixtures approach), correlating cytotoxicity scores for each cell line with the CCLE transcriptional data (RNA-seq), to reveal genes that are associated with resistance or sensitivity to NK cells. In addition, we applied genome-scale CRISPR-based gene editing screens in several solid tumor cell lines to interrogate, at a functional level, which genes regulate tumor cell response to NK cells. Based on these orthogonal studies, NK sensitive tumor cells tend to exhibit high levels of the NK cell-activating ligand B7-H6 (NCR3LG1); low levels of the inhibitory ligand HLA-E; microsatellite instability (MSI) status; high transcriptional signature for chromatin remodeling complexes and low antigen presentation machinery genes. Treatment with an HDAC inhibitor reduced the sensitivity of SW620 colon cancer cells, increased antigen presentation machinery, including HLA-E, and reduced B7-H6. Importantly, we observe that transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor resistance in clinical samples. Strikingly, comprehensive analysis of the CCLE transcriptional signatures revealed that cell lines with mesenchymal-like program tend to be more sensitive to NK cells treatment, compared with cell lines of epithelial-like program. Indeed, mesenchymal tumors tend to have lower expression of antigen presentation machinery in both CCLE and TCGA, suggesting a link between these two machieneries. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies. Citation Format: Michal Sheffer, Emily Lowry, Nicky Beelen, Minasri Borah, Suha Naffar-Abu Amara, Chris C. Mader, Jennifer Roth, Aviad Tsherniak, Olga Dashevsky, Sara Gandolfi, Samantha Bender, Jordan Bryan, Cong Zhu, Li Wang, Ricardo De-Matos Simoes, Channing Yu, Yiguo Hu, Olli Dufva, Marios Giannakis, Todd Golub, Rizwan Romee, Satu Mustjoki, Aedin C. Culhane, Lotte Wieten, Constantine S. Mitsiades. Landscape of molecular events regulating tumor cell responses to natural killer cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO041.

The use of microtechnique for studying cell division is well established (Begg & Ellis, 1979; Wadsworth, 1999; Zhang & Nicklas, 1999). The advantage of microinjection in cell division research is the timed delivery of a macromolecules at a particular stage of mitosis (for example, pre- vs postanaphase), which can circumvent the spindle assembly checkpoint (Hinchcliffe et al., 2016). Micromanipulation can be used to remove whole organelles, such as the centrosome or nucleus and examine the effects on cell division (Hinchcliffe et al., 2001; Hornick et al., 2011). The focus of this chapter is on methods for microinjection and micromanipulation of cultured mammalian cells. We describe pulling and shaping microneedles, as well as the imaging chambers we use. We also provide information on cell culture conditions, and imaging techniques used for our long-term observation studies, which allow cells to be followed on the order of several days.

The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity "Arg-like" SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an "Abl-like" low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases.

The curation and preservation of scientific data has long been recognized as an essential activity for the reproducibility of science and the advancement of knowledge. While investment into data curation for specific disciplines and at individual research institutions has advanced the ability to preserve research data products, data curation for big interdisciplinary science remains relatively unexplored terrain. To fill this lacunae, this article presents a case study of the data curation for the National Centers for Coastal Ocean Science (NCCOS) funded project “Understanding Coral Ecosystem Connectivity in the Gulf of Mexico-Pulley Ridge to the Florida Keys” undertaken from 2011 to 2018 by more than 30 researchers at several research institutions. The data curation process is described and a discussion of strengths, weaknesses and lessons learned is presented. Major conclusions from this case study include: the reimplementation of data repository infrastructure builds valuable institutional data curation knowledge but may not meet data curation standards and best practices; data from big interdisciplinary science can be considered as a special collection with the implication that metadata takes the form of a finding aid or catalog of datasets within the larger project context; and there are opportunities for data curators and librarians to synthesize and integrate results across disciplines and to create exhibits as stories that emerge from interdisciplinary big science. The substance of this article is based upon a poster presented at RDAP Summit 2019.

A comprehensive analysis of FDA approved drugs has shown that drug discovery through phenotypic screening yields a higher percentage of first-in-class compounds, compared to canonical target-based drug discovery. This has led to a renewed interest in phenotypic screening to identify novel mechanisms of action. The PRISM technology of adding a unique DNA barcode to cancer cell lines has been able to reduce the cost and time associated with phenotypic viability screening. Using the PRISM method, we profiled the cytotoxic activity of over 4,100 bioactive compounds in 578 barcoded cancer cell lines at one dose, followed by 8-point dose of 1100 active compounds. The use of genomically characterized cell lines from the Cancer Cell Line Encyclopedia (CCLE) allows us to identify baseline features associated with sensitivity to a particular compound. One of the compounds from the screen that we selected for follow up was BRD-K01353379, which killed 28 cancer cell lines of diverse lineages and showed a correlation between high expression of Ethanolamine Kinase 1 (ETNK1) and reduction of cell viability. Interestingly, BRD-3379 showed a similar PRISM cytotoxic profile to 2,3-DCPE, that was screened in our Repurposing effort. 2,3-DCPE is structurally similar to BRD-3379 and has been reported to induce cytotoxicity through upregulation of p21 and downregulation of Bcl-XL. To elucidate the mechanism of action of this compound we performed a genome-wide CRISPR-Cas9 resistance screen to identify molecular targets that are required for sensitivity to BRD-3379. Here, we found that loss of expression of the PRISM biomarker ETNK1 conferred resistance in a BRD-3379 sensitive cell line, JHH-7. Subsequently, we profiled metabolites in a cell line sensitive to BRD-3379 and found that the compound was phosphorylated. We confirmed in a biochemical assay that BRD-3379 is in fact a substrate of recombinant ETNK1, which phosphorylates the ethanolamine moiety of BRD-3379. Next, we transcriptionally profiled the effect of BRD-3379 by bulk RNAseq in a sensitive and non-sensitive cell line, as well as in a pool of 100 cell lines using single cell RNAseq [McFarland et al., manuscript in preparation]. Both bulk RNAseq and single cell transcriptional analysis showed that upregulation of both p21 and p57 is strongly associated with sensitivity to BRD-3379. This mechanism of action is similar to that of 2,3-DCPE. Here, we have demonstrated that by combining large-scale cell line viability screening, genomic characterization of these cell lines, genome wide CRISPR-Cas9 resistance screens, transcriptional profiling, and biochemical assays, we can facilitate rapid identification of molecular targets of small molecules and their respective mechanism of action by which they induce cytotoxicity. Citation Format: Kevin Larpenteur, Lucian de Waal, Li Wang, Mustafa Kocak, Jordan Bryan, Samantha Bender, Sulyman Barkho, Matt Kukurugya, Amy Goodale, Brenton Paolella, Min Cho, James McFarland, Michael Rothberg, Cong Zhu, Colin Trepiccio, Aviad Tsherniak, David Root, Bryson Bennett, Federica Piccioni, Josh Bittker, James Rong, Christopher Mader, David Stokoe, Ari Firestone, Todd R Golub, Jennifer Roth. Large scale phenotypic screening using PRISM, integrated with functional genomic screening and transcriptional profiling accelerates target identification of cytotoxic small molecules [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C022. doi:10.1158/1535-7163.TARG-19-C022

Intratumoral heterogeneity impacts both tumor progression and drug sensitivity. While intratumoral genetic heterogeneity has been extensively described, there is still little understanding of the extent of heterogeneity in drug sensitivity within a single tumor, the intra tumor dynamics of different cell populations during anti-cancer compound treatment, and the role of non-cell autonomous mechanism in drug resistance. The objective of this study is to characterize heterogeneity in drug sensitivity within a model for human breast cancer, identify interactions between clonal population influencing drug sensitivity, and use this to model the dynamics of sub populations in a tumor during anti-cancer compound treatment. We have characterized 31 subclonal cell populations (SCPs) that were generated by single cell cloning of the triple-negative breast cancer cell line MDA-MB-468. These SCPs display considerable phenotypic and molecular heterogeneity with respect to morphology, proliferation rate, RNA expression and tumorigenicity in mice. To examine the extent of heterogeneity in drug sensitivity, we designed and screened a 200-small molecule compound library in 24 SCPs and the parental cell line. We observed considerable heterogeneity in drug sensitivity and selected 36 compounds for validation in the SCPs that expressed the greatest range of sensitivity (determined by IC50). To investigate whether interactions between clonal population can influence drug sensitivity heterogeneously responsive subclonal cell lines were transduced with lentiviruses encoding for the H2B-GFP or H2B-RFP fusion protein and then cocultured in the absence or presence of different compounds. We observed shifts in drug sensitivity for SCPs that were cocultured with differentially responsive clones. Examples include SCPs 19 and 27 two highly sensitive populations that were protected by the presence of less sensitive SCPs when under gemcitabine treatment, resulting in IC50-values that were 20-fold higher then when cultured alone. RNA-sequencing and RPPA analysis will be used to characterize this non-cell autonomous mechanism of resistance and the relationship with identified transcriptional programs or genes which expression correlates with drug sensitivity. We are also generating and will evaluate predictive models for drug combination using the SCP drug sensitivity profiles. The results of this study show that there is considerable heterogeneity in drug sensitivity among clonal subpopulations derived from a single cancer cell line. Ongoing experiments will provide insight into the effect of drug combinations and unique treatment schedules on distinct tumor cell subpopulations and drug sensitivity within heterogenous mixtures. Moreover, we will be able to test for and investigate non-cell autonomous modes of drug resistance, which may guide the development of new drug combination treatments or schedules. Citation Format: Chandler M Friend, Joan S Brugge, Hendrik J Kuiken, Steven M Corsello, Christopher C Mader. Characterization of intratumoral heterogeneity in drug sensitivity and non-cell autonomous mechanisms of drug resistance using subclonal cell populations derived from a single breast cancer cell line [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A132. doi:10.1158/1535-7163.TARG-19-A132

KEAP1-mutant non-small cell lung cancer is a high prevalence indication that responds poorly to conventional chemotherapy owing to constitutive activation of NRF2 and its associated drug metabolism target genes. Using an approach to identify compounds that selectively kill cancer cell lines in a biomarker-driven manner, we identify BRD-K19050021 (K1905), a compound displaying strong toxicity in cells expressing CYP4F11 - a cytochrome p450 family member and NRF2 transcriptional target. Using genome-wide pooled CRISPR screens, we show that CYP4F11 activity is necessary and sufficient for response to K1905, and in acquired resistance models developed from multiple cell lines, we show a minor subpopulation of cells mechanistically converge on suppressed CYP4F11 expression to bypass K1905 response. CYP4F11 converts K1905 from a prodrug into a covalent active metabolite that alkylates several cellular targets, triggering all three canonical arms of the unfolded protein response pathway and culminating in cell death. We propose that CYP4F11 and similar metabolic enzyme activities promoted by oncogenic drivers represent a unique opportunity to restrict prodrug activation within tumors, provided that normal tissue expression of such prodrug-converting enzymes do not diminish therapeutic index. Citation Format: Aaron Boudreau, Jennifer Roth, James Rong, Niclas Olsson, Chris Mader, Jordan Byran, Jordan Rossen, Li Wang, Kevin Larpenteur, Amy Goodale, Colin Trepicchio, Samantha Bender, Aviad Tsherniak, Aravind Subramanian, Mustafa Kocak, Federica Piccioni, Josh Bittker, Cong Zhu, Frank Li, Nick Eriksson, Daphne Koller, Fiona McAllister, Todd Golub, Jeff Settleman, Ari Firestone, David Stokoe. An oncogene-linked prodrug strategy in lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B121. doi:10.1158/1535-7163.TARG-19-B121

Intratumoral heterogeneity can have profound effects on tumor progression and drug resistance. While tumor cell heterogeneity has been described at many levels, there is a poor understanding of the extent of heterogeneity in drug sensitivity within a single tumor, the dynamics of tumor cell subpopulations during treatment with anti-cancer agents and non-cell autonomous mechanisms of drug resistance. The overall objective of this study is to characterize heterogeneity in drug sensitivity within a model for human breast cancer and to model and evaluate the effect of drug combinations on the dynamics of subpopulations within heterogeneous mixtures. We have characterized 31 subclonal cell populations (SCPs) that were generated through single cell cloning of the triple-negative breast cancer cell line MDA-MB-468. These SCPs display considerable heterogeneity with respect to morphology, proliferation rate, colony formation in soft agar and tumorigenicity in mice. To examine the extent of heterogeneity in drug sensitivity, we designed and screened a 200-small molecule compound library in 24 SCPs and the parental cell line. We observed considerable heterogeneity in drug sensitivity and selected 36 compounds for validation, including GPX4 inhibitor ML-210 and gemcitabine, to which the SCPs displayed 12-fold and 150-fold differences in the EC50, respectively. The results of these validation experiments were used to identify transcriptional programs and individual genes which expression correlates with drug sensitivity. We have generated and will evaluate predictive models for drug combination using the SCP drug sensitivity profiles. To test the effect of individual drugs and combinations, as well as different treatment schedules on heterogeneous populations, we will examine the dynamics of individual SCPs within mixed cultures. To this end, we have transduced 22 SCPs with CloneTracer lentiviruses, each encoding for a unique DNA barcode. The distribution of these unique barcodes can be determined by next generation sequencing. Previously, we have found that propagation of a 22 SCP mixture results in reproducible changes in the composition of mixed monolayer cultures with approximately half of the SCPs having a relative abundance of 1% each in the mixed population following propagation for four months. The results of this study show that there is considerable heterogeneity in drug sensitivity among clonal subpopulations derived from a single cancer cell line. Ongoing experiments will provide insight into the effect of drug combinations and unique treatment schedules on distinct tumor cell subpopulations within heterogenous mixtures. Moreover, using this panel of SCPs, we will be able to test for and investigate non-cell autonomous modes of drug resistance, which may guide the development of new drug combinations. Citation Format: Hendrik J. Kuiken, Chandler M. Friend, Steven M. Corsello, Christopher C. Mader, Joan S. Brugge. Characterization of intratumoral heterogeneity in drug sensitivity and modeling of drug combination effects using subclonal cell populations derived from a single breast cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3775.

The complexity of biology makes defining a single model system that recapitulates human disease a nearly impossible task. To overcome this inherent limitation, screening of many different systems across multiple measurement parameters enables the generation of response signatures or profiles that are more predictive and robust than measurements in single systems alone. The application of signature based discovery has included early efforts such as NCI-60, but has been limited by the amount of additional dimensional data and technical and logistical challenges that prevented collection of large scale profiles. With the recent development of large datasets of genomic characterization and annotations of cell lines, and the development of new technologies to enable measurements of profiles across hundreds of cellular systems in a cost and time efficient manner, it is now possible to routinely collect multidimensional profiles during the discovery process. Similarly, advancements in computational approaches such as the Connectivity Map allow for sophisticated queries across various profile based datasets to find correlations such as target identification and mechanisms of action. Taken together, multidimensional profile based screening can provide a wealth of information and actionable hypotheses at multiple stages of the drug development process.

SummaryThe role of centrosomes and centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles [1–3]. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays—termed an amphiaster (“a star on both sides” [4])—that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB) [5]. Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC reassembled, and, prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ∼35% of aMTOCs failed to split and separate before NEB, and these entered mitosis with persistent monastral spindles. Chromatin-associated RAN-GTP—the small GTPase Ran in its GTP bound state—could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but, in its absence, the fidelity of bipolar spindle assembly is highly compromised.

Invasive carcinoma cells use specialized actin polymerization–driven protrusions called invadopodia to degrade and possibly invade through the extracellular matrix (ECM) during metastasis. Phosphorylation of the invadopodium protein cortactin is a master switch that activates invadopodium maturation and function. Cortactin was originally identified as a hyperphosphorylated protein in v-Src–transformed cells, but the kinase or kinases that are directly responsible for cortactin phosphorylation in invadopodia remain unknown. In this study, we provide evidence that the Abl-related nonreceptor tyrosine kinase Arg mediates epidermal growth factor (EGF)–induced cortactin phosphorylation, triggering actin polymerization in invadopodia, ECM degradation, and matrix proteolysis–dependent tumor cell invasion. Both Src and Arg localize to invadopodia and are required for EGF-induced actin polymerization. Notably, Arg overexpression in Src knockdown cells can partially rescue actin polymerization in invadopodia while Src overexpression cannot compensate for loss of Arg, arguing that Src indirectly regulates invadopodium maturation through Arg activation. Our findings suggest a novel mechanism by which an EGFR–Src–Arg–cortactin pathway mediates functional maturation of invadopodia and breast cancer cell invasion. Furthermore, they identify Arg as a novel mediator of invadopodia function and a candidate therapeutic target to inhibit tumor invasion in vivo. Cancer Res; 71(5); 1730–41. ©2011 AACR.

Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells. The mechanisms regulating invadopodium assembly and maturation are not understood. We have dissected the stages of invadopodium assembly and maturation and show that invadopodia use cortactin phosphorylation as a master switch during these processes. In particular, cortactin phosphorylation was found to regulate cofilin and Arp2/3 complex–dependent actin polymerization. Cortactin directly binds cofilin and inhibits its severing activity. Cortactin phosphorylation is required to release this inhibition so cofilin can sever actin filaments to create barbed ends at invadopodia to support Arp2/3-dependent actin polymerization. After barbed end formation, cortactin is dephosphorylated, which blocks cofilin severing activity thereby stabilizing invadopodia. These findings identify novel mechanisms for actin polymerization in the invadopodia of metastatic carcinoma cells and define four distinct stages of invadopodium assembly and maturation consisting of invadopodium precursor formation, actin polymerization, stabilization, and matrix degradation.

New therapies are desperately needed for patients with advanced cancer, but making safe and efficacious drugs remains expensive and time consuming. Meanwhile, multiple non-oncology drugs have been successfully repurposed for cancer, including thalidomide for multiple myeloma and aspirin for prevention of colorectal cancer. While the benefits of repurposing are clear, successes to date have been largely serendipitous. We have created two foundational tools to identify drug repurposing opportunities at a much greater scale: a new world-class screening library of more than 4,000 drugs/clinical candidates and a multiplexed method to rapidly conduct cellular viability assays across hundreds of cell lines. PRISM, a recently developed high-throughput assay, employs DNA-barcoded cell lines to enable rapid testing of many drugs against pools of cancer cell lines. We have tested 4,100 compounds against 578 genomically characterized cancer cell lines using PRISM. Our experiment is among the largest cell line screens ever undertaken. Global analysis has revealed multiple strong clusters of active drugs, including vitamin D agonists, bromodomain inhibitors, and statins. In addition, many novel and established drug-biomarker relationships were identified, including alkylating agent response and low MGMT expression, BRAF inhibitor response and BRAF mutation, and MDM2 inhibitor response and TP53 wild-type status. Surprisingly, more than 100 non-oncology drugs unexpectedly killed multiple cancer cell lines, making them attractive repurposing candidates. For example, the FDA-approved phosphodiesterase inhibitor anagrelide selectively kills PDE3A-high lung cancer and melanoma cell lines. Confirmatory experiments are underway to validate screening results, evaluate putative biomarkers, and test drug activity in preclinical models. Importantly, this approach is expected to enable rapid initiation of clinical trials, dramatically accelerating patient access to potential new therapies. Citation Format: Steven M. Corsello, Christopher C. Mader, Jordan Bryan, Jennifer Roth, David Peck, John Davis, Samantha Bender, Li Wang, Alice Wang, Joshua Bittker, Francisca Vazquez, Aravind Subramanian, Aviad Tsherniak, Todd R. Golub. Accelerating drug repurposing for cancer therapy using multiplexed viability assays [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 4025. doi:10.1158/1538-7445.AM2017-4025

The National Institutes of Health Library of Integrated Network-based Cellular Signatures (LINCS) program is generating extensive multidimensional data sets, including biochemical, genome-wide transcriptional, and phenotypic cellular response signatures to a variety of small-molecule and genetic perturbations with the goal of creating a sustainable, widely applicable, and readily accessible systems biology knowledge resource. Integration and analysis of diverse LINCS data sets depend on the availability of sufficient metadata to describe the assays and screening results and on their syntactic, structural, and semantic consistency. Here we report metadata specifications for the most important molecular and cellular components and recommend them for adoption beyond the LINCS project. We focus on the minimum required information to model LINCS assays and results based on a number of use cases, and we recommend controlled terminologies and ontologies to annotate assays with syntactic consistency and semantic integrity. We also report specifications for a simple annotation format (SAF) to describe assays and screening results based on our metadata specifications with explicit controlled vocabularies. SAF specifically serves to programmatically access and exchange LINCS data as a prerequisite for a distributed information management infrastructure. We applied the metadata specifications to annotate large numbers of LINCS cell lines, proteins, and small molecules. The resources generated and presented here are freely available.

We report the development of a powerful analytical method that utilizes a tilted elastomeric pyramidal pen array in the context of a scanning probe lithography experiment to rapidly prepare libraries having as many as 25 million features over large areas with a range of feature sizes from the nano- to microscale. This technique can be used to probe important chemical and biological processes, opening up the field of nanocombinatorics. In a proof-of-concept investigation of mesenchymal stem cell (MSC) differentiation, combinatorial patterns first enabled a rapid and systematic screening of MSC adhesion, as a function of feature size, while uniform patterns were used to study differentiation with statistically significant sample sizes. Without media containing osteogenic-inducing chemical cues, cells cultured on nanopatterned fibronectin substrates direct MSC differentiation towards osteogenic fates when compared to nonpatterned fibronectin substrates. This powerful and versatile approach enables studies of many systems spanning biology, chemistry, and engineering areas.

Cortactin phosphorylation induces recruitment of the sodium-hydrogen exchanger NHE1 to invadopodia, resulting in pH changes that regulate cortactin-cofilin binding and invadopodium dynamics., Invadopodia are invasive protrusions with proteolytic activity uniquely found in tumor cells. Cortactin phosphorylation is a key step during invadopodia maturation, regulating Nck1 binding and cofilin activity. The precise mechanism of cortactin-dependent cofilin regulation and the roles of this pathway in invadopodia maturation and cell invasion are not fully understood. We provide evidence that cortactin–cofilin binding is regulated by local pH changes at invadopodia that are mediated by the sodium–hydrogen exchanger NHE1. Furthermore, cortactin tyrosine phosphorylation mediates the recruitment of NHE1 to the invadopodium compartment, where it locally increases the pH to cause the release of cofilin from cortactin. We show that this mechanism involving cortactin phosphorylation, local pH increase, and cofilin activation regulates the dynamic cycles of invadopodium protrusion and retraction and is essential for cell invasion in 3D. Together, these findings identify a novel pH-dependent regulation of cell invasion.

Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells enriched in proteins that regulate actin polymerization. The on–off regulatory switch that initiates actin polymerization in invadopodia requires phosphorylation of tyrosine residues 421, 466, and 482 on cortactin. However, it is unknown which of these cortactin tyrosine phosphorylation sites control actin polymerization. We investigated the contribution of individual tyrosine phosphorylation sites (421, 466, and 482) on cortactin to the regulation of actin polymerization in invadopodia. We provide evidence that the phosphorylation of tyrosines 421 and 466, but not 482, is required for the generation of free actin barbed ends in invadopodia. In addition, these same phosphotyrosines are important for Nck1 recruitment to invadopodia via its SH2 domain, for the direct binding of Nck1 to cortactin in vitro, and for the FRET interaction between Nck1 and cortactin in invadopodia. Furthermore, matrix proteolysis-dependent tumor cell invasion is dramatically inhibited in cells expressing a mutation in phosphotyrosine 421 or 466. Together, these results identify phosphorylation of tyrosines 421 and 466 on cortactin as the crucial residues that regulate Nck1-dependent actin polymerization in invadopodia and tumor cell invasion, and suggest that specifically blocking either tyrosine 421 or 466 phosphorylation might be effective at inhibiting tumor cell invasion in vivo.

The molecular mechanisms by which the Abelson (Abl) or Abl-related gene (Arg) kinases interface with the actin polymerization machinery to promote cell edge protrusions during cell–matrix adhesion are unclear. In this study, we show that interactions between Arg and the Arp2/3 complex regulator cortactin are essential to mediate actin-based cell edge protrusion during fibroblast adhesion to fibronectin. Arg-deficient and cortactin knockdown fibroblasts exhibit similar defects in adhesion-dependent cell edge protrusion, which can be restored via reexpression of Arg and cortactin. Arg interacts with cortactin via both binding and catalytic events. The cortactin Src homology (SH) 3 domain binds to a Pro-rich motif in the Arg C terminus. Arg mediates adhesion-dependent phosphorylation of cortactin, creating an additional binding site for the Arg SH2 domain. Mutation of residues that mediate Arg–cortactin interactions abrogate the abilities of both proteins to support protrusions, and the Nck adapter, which binds phosphocortactin, is also required. These results demonstrate that interactions between Arg, cortactin, and Nck1 are critical to promote adhesion-dependent cell edge protrusions.