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Supplementary Table 1 showing reagents and antibodies used in the study.

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Homologous recombination (HR)-deficient cancers are sensitive to poly-ADP ribose polymerase inhibitors (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive TP53−/− and BRCA1−/− epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison with a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained preexisting drug-tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical patient with BRCA1-mutant HGSC with acquired PARPi resistance was observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance.Significance:This study shows that BRCA1-deficient cells can give rise to multiple genomically and functionally heterogenous PARPi-resistant clones, which are associated with various vulnerabilities that can be targeted in a mechanism-specific manner.

Suppl. F1: On-target activity and toxicity of CX-6258 Suppl. F2: On-target activity of CX-6258 and siRNA KD of Haspin Suppl. F3: Effects of CX-6258 on cell-cycle and cGAS recruitment Suppl. F4: In vivo effects of CX-6258

Suppl. Table 1: Compounds used in drug screen

Supplementary Table 2 showing the positive and negative GSEA results for each cell line.

Patients with melanoma resistant to RAF/MEK inhibitors (RMi) are frequently resistant to other therapies, such as immune checkpoint inhibitors (ICI), and individuals succumb to their disease. New drugs that control tumor growth and favorably modulate the immune environment are therefore needed. We report that the small-molecule CX-6258 has potent activity against both RMi-sensitive (RMS) and -resistant (RMR) melanoma cell lines. Haspin kinase (HASPIN) was identified as a target of CX-6258. HASPIN inhibition resulted in reduced proliferation, frequent formation of micronuclei, recruitment of cGAS, and activation of the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway. In murine models, CX-6258 induced a potent cGAS-dependent type-I IFN response in tumor cells, increased IFNγ-producing CD8+ T cells, and reduced Treg frequency in vivo. HASPIN was more strongly expressed in malignant compared with healthy tissue and its inhibition by CX-6258 had minimal toxicity in ex vivo–expanded human tumor-infiltrating lymphocytes (TIL), proliferating TILs, and in vitro differentiated neurons, suggesting a potential therapeutic index for anticancer therapy. Furthermore, the activity of CX-6258 was validated in several Ewing sarcoma and multiple myeloma cell lines. Thus, HASPIN inhibition may overcome drug resistance in melanoma, modulate the immune environment, and target a vulnerability in different cancer lineages.Significance:HASPIN inhibition by CX-6258 is a novel and potent strategy for RAF/MEK inhibitor–resistant melanoma and potentially other tumor types. HASPIN inhibition has direct antitumor activity and induces a favorable immune microenvironment.

Methods and Suppl. Figure Legends

Homologous recombination (HR)-deficient cancers are sensitive to poly-ADP ribose polymerase inhibitors (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive TP53−/− and BRCA1−/− epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison with a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained preexisting drug-tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical patient with BRCA1-mutant HGSC with acquired PARPi resistance was observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance. Significance: This study shows that BRCA1-deficient cells can give rise to multiple genomically and functionally heterogenous PARPi-resistant clones, which are associated with various vulnerabilities that can be targeted in a mechanism-specific manner.

Cyclin-dependent kinase 2 (CDK2) is a potential therapeutic target for the treatment of cancer. Development of CDK2 inhibitors has been extremely challenging as its ATP-binding site shares high similarity with CDK1, a related kinase whose inhibition causes toxic effects. Here, we report the development of TMX-2172, a heterobifunctional CDK2 degrader with degradation selectivity for CDK2 and CDK5 over not only CDK1, but transcriptional CDKs (CDK7 and CDK9) and cell cycle CDKs (CDK4 and CDK6) as well. In addition, we demonstrate that antiproliferative activity in ovarian cancer cells (OVCAR8) depends on CDK2 degradation and correlates with high expression of cyclin E1 (CCNE1), which functions as a regulatory subunit of CDK2. Collectively, our work provides evidence that TMX-2172 represents a lead for further development and that CDK2 degradation is a potentially valuable therapeutic strategy in ovarian and other cancers that overexpress CCNE1.

Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking. We identified the innate immune kinase TANK-binding kinase 1 (TBK1) as a candidate immune evasion gene in a pooled genetic screen. Using a suite of genetic and pharmacologic tools across multiple experimental model systems, we confirm a role for TBK1 as an immune evasion gene. Targeting TBK1 enhances response to PD-1 blockade by lowering the cytotoxicity threshold to effector cytokines (TNFα/IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumor models. Cancer cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNFα/IFNγ in a JAK/STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is a novel and effective strategy to overcome resistance to cancer immunotherapy. Citation Format: Yi Sun, Or-Yam Revach, Seth Anderson, Anne Jenney, Caitlin E. Mills, Payal Tiwari, Robert T. Manguso, Russell William Jenkins. Targeting TBK1 to overcome resistance to cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3285.

High-throughput measurement of cells perturbed using libraries of small molecules, gene knockouts, or different microenvironmental factors is a key step in functional genomics and pre-clinical drug discovery. However, it remains difficult to perform accurate single-cell assays in 384-well plates, limiting many studies to well-average measurements (e.g. CellTiter-Glo®). Here we describe a public domain “Dye Drop” method that uses sequential density displacement and microscopy to perform multi-step assays on living cells. We use Dye Drop cell viability and DNA replication assays followed by immunofluorescence imaging to collect single-cell dose-response data for 67 investigational and clinical-grade small molecules in 58 breast cancer cell lines. By separating the cytostatic and cytotoxic effects of drugs computationally, we uncover unexpected relationships between the two. Dye Drop is rapid, reproducible, customizable, and compatible with manual or automated laboratory equipment. Dye Drop improves the tradeoff between data content and cost, enabling the collection of information-rich perturbagen-response datasets.

High-throughput measurement of cells perturbed using libraries of small molecules, gene knockouts, or different microenvironmental factors is a key step in functional genomics and pre-clinical drug discovery. However, it remains difficult to perform accurate single-cell assays in 384-well plates, limiting many studies to well-average measurements (e.g., CellTiter-Glo®). Here we describe a public domain Dye Drop method that uses sequential density displacement and microscopy to perform multi-step assays on living cells. We use Dye Drop cell viability and DNA replication assays followed by immunofluorescence imaging to collect single-cell dose-response data for 67 investigational and clinical-grade small molecules in 58 breast cancer cell lines. By separating the cytostatic and cytotoxic effects of drugs computationally, we uncover unexpected relationships between the two. Dye Drop is rapid, reproducible, customizable, and compatible with manual or automated laboratory equipment. Dye Drop improves the tradeoff between data content and cost, enabling the collection of information-rich perturbagen-response datasets.

SUMMARYThe phenotype of a cell and its underlying molecular state is strongly influenced by extracellular signals, including growth factors, hormones, and extracellular matrix. While these signals are normally tightly controlled, their dysregulation leads to phenotypic and molecular states associated with diverse diseases. To develop a detailed understanding of the linkage between molecular and phenotypic changes, we generated a comprehensive dataset that catalogs the transcriptional, proteomic, epigenomic and phenotypic responses of MCF10A mammary epithelial cells after exposure to the ligands EGF, HGF, OSM, IFNG, TGFB and BMP2. Systematic assessment of the molecular and cellular phenotypes induced by these ligands comprise the LINCS Microenvironment (ME) perturbation dataset, which has been curated and made publicly available for community-wide analysis and development of novel computational methods (synapse.org/LINCS_MCF10A). In illustrative analyses, we demonstrate how this dataset can be used to discover functionally related molecular features linked to specific cellular phenotypes.

Deubiquitinating enzymes (DUBs), ~100 of which are found in human cells, are proteases that remove ubiquitin conjugates from proteins, thereby regulating protein turnover. They are involved in a wide range of cellular activities and are emerging therapeutic targets for cancer and other diseases. Drugs targeting USP1 and USP30 are in clinical development for cancer and kidney disease respectively. However, the majority of substrates and pathways regulated by DUBs remain unknown, impeding efforts to prioritize specific enzymes for research and drug development. To assemble a knowledgebase of DUB activities, co-dependent genes, and substrates, we combined targeted experiments using CRISPR libraries and inhibitors with systematic mining of functional genomic databases. Analysis of the Dependency Map, Connectivity Map, Cancer Cell Line Encyclopedia, and multiple protein-protein interaction databases yielded specific hypotheses about DUB function, a subset of which were confirmed in follow-on experiments. The data in this paper are browsable online in a newly developed DUB Portal and promise to improve understanding of DUBs as a family as well as the activities of incompletely characterized DUBs (e.g. USPL1 and USP32) and those already targeted with investigational cancer therapeutics (e.g. USP14, UCHL5, and USP7).

The recent introduction of small molecule inhibitors of cyclin-dependent kinases (CDK) 4/6 to the clinic has improved the treatment of hormone receptor positive breast cancer, and shown promise in other malignancies. The three clinically used CDK4/6 inhibitors, palbociclib, ribociclib, and abemaciclib, are reported to be broadly similar although recent data suggest that abemaciclib has distinct single-agent activity in patients and a unique adverse effects profile. Key questions are: How do these drugs differ at the molecular level? Should such differences inform their use in the clinic? Can these three agents be used interchangeably or should patient stratification differ between them? We use molecular and functional profiling by mRNA sequencing, mass spectrometry-based proteomics, and GR-based dose-response assays to obtain complementary views of the mechanisms of action of CDK4/6 inhibitors. We show that abemaciclib, but not ribociclib or palbociclib, is a potent inhibitor of kinases other than CDK4/6, including CDK1/Cyclin B, which appears to cause arrest in the G2 phase of the cell cycle, and CDK2/Cyclin E/A, which is implicated in resistance to palbociclib. We show that inhibition of these additional targets is accessible in a xenograft model. Whereas ribociclib and palbociclib induce cytostasis, and cells adapt to these drugs within 2-3 days of exposure, abemaciclib induces cell death and durably blocks cell proliferation. Abemaciclib is active even in retinoblastoma protein (pRb)-deficient cells in which CDK4/6 inhibition by palbociclib or ribociclib is completely ineffective. The degree of polypharmacology of small molecule drugs is increasingly viewed as an important consideration in their design, with implications for efficacy, toxicity, and acquired resistance. In the case of CDK4/6 inhibitors, we propose that abemaciclib polypharmacology elicits unique molecular responses. More generally, we propose that multi-omic approaches are required to fully elucidate the spectrum of targets relevant to drug action in tumor cells. We expect such understanding to assist in stratifying patient populations and ordering sequential therapies when resistance arises. Citation Format: Mills CE, Hafner M, Subramanian K, Chen C, Chung M, Boswell SA, Everley RA, Walmsley CS, Juric D, Sorger PK. Omics profiling of CDK4/6 inhibitors reveals functionally important secondary targets of abemaciclib [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD1-12.

Proliferation is a fundamental trait of cancer cells, but its properties and spatial organization in tumours are poorly characterized. Here we use highly multiplexed tissue imaging to perform single-cell quantification of cell cycle regulators and then develop robust, multivariate, proliferation metrics. Across diverse cancers, proliferative architecture is organized at two spatial scales: large domains, and smaller niches enriched for specific immune lineages. Some tumour cells express cell cycle regulators in the (canonical) patterns expected of freely growing cells, a phenomenon we refer to as 'cell cycle coherence'. By contrast, the cell cycles of other tumour cell populations are skewed towards specific phases or exhibit non-canonical (incoherent) marker combinations. Coherence varies across space, with changes in oncogene activity and therapeutic intervention, and is associated with aggressive tumour behaviour. Thus, multivariate measures from high-plex tissue images capture clinically significant features of cancer proliferation, a fundamental step in enabling more precise use of anti-cancer therapies.

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS), current Version 1.0, is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

We performed quantitative proteomics on 60 human-derived breast cancer cell lines to a depth of ~13,000 proteins. The resulting high-throughput datasets were assessed for quality and reproducibility. We used the datasets to identify and characterize the subtypes of breast cancer and showed that they conform to known transcriptional subtypes, revealing that molecular subtypes are preserved even in under-sampled protein feature sets. All datasets are freely available as public resources on the LINCS portal. We anticipate that these datasets, either in isolation or in combination with complimentary measurements such as genomics, transcriptomics and phosphoproteomics, can be mined for the purpose of predicting drug response, informing cell line specific context in models of signalling pathways, and identifying markers of sensitivity or resistance to therapeutics.

Homologous recombination (HR)-deficient cancers are sensitive to inhibitors of Poly-ADP Ribose Polymerase (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive, TP53−/− and BRCA1−/− epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison to a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained pre-existing drug tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical BRCA1-mutant HGSC patient with acquired PARPi resistance were observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance.

Malignant abdominal fluid (ascites) frequently develops in women with advanced high-grade serous ovarian cancer (HGSOC) and is associated with drug resistance and a poor prognosis1. To comprehensively characterize the HGSOC ascites ecosystem, we used single-cell RNA sequencing to profile ~11,000 cells from 22 ascites specimens from 11 patients with HGSOC. We found significant inter-patient variability in the composition and functional programs of ascites cells, including immunomodulatory fibroblast sub-populations and dichotomous macrophage populations. We found that the previously described immunoreactive and mesenchymal subtypes of HGSOC, which have prognostic implications, reflect the abundance of immune infiltrates and fibroblasts rather than distinct subsets of malignant cells2. Malignant cell variability was partly explained by heterogeneous copy number alteration patterns or expression of a stemness program. Malignant cells shared expression of inflammatory programs that were largely recapitulated in single-cell RNA sequencing of ~35,000 cells from additionally collected samples, including three ascites, two primary HGSOC tumors and three patient ascites-derived xenograft models. Inhibition of the JAK/STAT pathway, which was expressed in both malignant cells and cancer-associated fibroblasts, had potent anti-tumor activity in primary short-term cultures and patient-derived xenograft models. Our work contributes to resolving the HSGOC landscape3-5 and provides a resource for the development of novel therapeutic approaches.

The Library of Integrated Network-Based Cellular Signatures (LINCS) is an NIH Common Fund program that catalogs how human cells globally respond to chemical, genetic, and disease perturbations. Resources generated by LINCS include experimental and computational methods, visualization tools, molecular and imaging data, and signatures. By assembling an integrated picture of the range of responses of human cells exposed to many perturbations, the LINCS program aims to better understand human disease and to advance the development of new therapies. Perturbations under study include drugs, genetic perturbations, tissue micro-environments, antibodies, and disease-causing mutations. Responses to perturbations are measured by transcript profiling, mass spectrometry, cell imaging, and biochemical methods, among other assays. The LINCS program focuses on cellular physiology shared among tissues and cell types relevant to an array of diseases, including cancer, heart disease, and neurodegenerative disorders. This Perspective describes LINCS technologies, datasets, tools, and approaches to data accessibility and reusability.

Drug response screens on panels of cell lines aimed at identifying markers of sensitivity or resistance have been limited in their successes. Unfortunately, the recent release of many such studies has been accompanied by concerns surrounding reproducibility. Since then, several publications have addressed these concerns by pointing out sources of variability and by suggesting better experimental methods as well as more robust analytical approaches. In the presented profiling effort, we integrated the latest advances in drug response measurement and focused on data diversity and quality rather than on breadth. We selected 32 breast cancer cell lines with a strong bias towards triple negative lines as well as 4 cell lines established from relevant patient-derived xenografts. We used high content microscopy to assay the phenotypic responses of the cell lines to a panel of 34 drugs made up largely of kinase inhibitors currently in the clinic along with some standard of care chemotherapeutics. The microscopy based drug response assay allowed us to measure drug potency, and to quantify the efficacy of the drugs in terms of growth inhibition and cell death. For the same cell lines, we used RNAseq to measure basal mRNA expression levels and shotgun mass spectrometry to measure endogenous protein levels. The completeness and controlled conditions under which these data sets were collected provide confidence in their integration. The complementarity of these multi-omics data has allowed us to address questions about the landscape of, particularly triple negative, breast cancer cell lines. Such questions include: where do the patient-derived lines lay among the established cell lines? and how different are the landscapes defined by drug response phenotypes, mRNA expression, and protein levels? We used network-based algorithms to identify eigenstates of signaling pathways related to genomic events, and further explored these states in the TCGA data. At the level of drug response, we have focused on important questions related to the clinical use of kinase inhibitors. In particular, we compared various CDK inhibitors in an effort to identify markers that are informative of response potency and efficacy. We have also looked at variability of the responses of the cell lines studied to multiple PI3K inhibitors that either target specific isoforms or all isoforms. Overall the data set that has been collected is a valuable resource for understanding drug response in triple negative breast cancer, and the transcriptomic and proteomic factors that influence it. Citation Format: Mills CE, Hafner MA, Sorger PK. Integration of transcriptomic, proteomic and drug response data in triple negative breast cancer cell lines and PDX models [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-08-02.

Patients with melanoma resistant to RAF/MEK inhibitors (RMi) are frequently resistant to other therapies, such as immune checkpoint inhibitors (ICI), and individuals succumb to their disease. New drugs that control tumor growth and favorably modulate the immune environment are therefore needed. We report that the small-molecule CX-6258 has potent activity against both RMi-sensitive (RMS) and -resistant (RMR) melanoma cell lines. Haspin kinase (HASPIN) was identified as a target of CX-6258. HASPIN inhibition resulted in reduced proliferation, frequent formation of micronuclei, recruitment of cGAS, and activation of the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway. In murine models, CX-6258 induced a potent cGAS-dependent type-I IFN response in tumor cells, increased IFNγ-producing CD8+ T cells, and reduced Treg frequency in vivo. HASPIN was more strongly expressed in malignant compared with healthy tissue and its inhibition by CX-6258 had minimal toxicity in ex vivo–expanded human tumor-infiltrating lymphocytes (TIL), proliferating TILs, and in vitro differentiated neurons, suggesting a potential therapeutic index for anticancer therapy. Furthermore, the activity of CX-6258 was validated in several Ewing sarcoma and multiple myeloma cell lines. Thus, HASPIN inhibition may overcome drug resistance in melanoma, modulate the immune environment, and target a vulnerability in different cancer lineages. Significance: HASPIN inhibition by CX-6258 is a novel and potent strategy for RAF/MEK inhibitor–resistant melanoma and potentially other tumor types. HASPIN inhibition has direct antitumor activity and induces a favorable immune microenvironment.

We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS) is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.

Frequent mutation of genes in the PI3K/AKT/mTOR signaling pathway in human cancers has stimulated large investments in therapeutic drugs but clinical successes have been rare. As a result, many cancers with high PI3K pathway activity such as triple-negative breast cancer (TNBC) are still treated primarily with conventional chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors, we find that one drug, Torin2, is unusually effective because it inhibits both mTOR and PI3K-like kinases (PIKKs). In contrast to mTOR-selective inhibitors, Torin2 exploits dependencies on several kinases for progression of S-phase and for cell cycle checkpoints, thereby causing accumulation of single-stranded DNA and death by replication catastrophe or mitotic failure. Thus, Torin2 and its analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors.

Doublecortin-like kinase 1 (DCLK1) is critical for neurogenesis, but overexpression is also observed in multiple cancers and is associated with poor prognosis. Nevertheless, the function of DCLK1 in cancer, especially the context-dependent functions, are poorly understood. We present a "toolkit" that includes the DCLK1 inhibitor DCLK1-IN-1, a complementary DCLK1-IN-1-resistant mutation G532A, and kinase dead mutants D511N and D533N, which can be used to investigate signaling pathways regulated by DCLK1. Using a cancer cell line engineered to be DCLK1 dependent for growth and cell migration, we show that this toolkit can be used to discover associations between DCLK1 kinase activity and biological processes. In particular, we show an association between DCLK1 and RNA processing, including the identification of CDK11 as a potential substrate of DCLK1 using phosphoproteomics.

Tumor/T-cell co-cultures in combination with large-scale genome-editing tools emerged as powerful tools for discovery of drivers of resistance to immunotherapies. Traditionally, chromium or lactate-dehydrogenase release and/or target cell viability counts before and after co-culture treatment are used as surrogate endpoint assays to estimate the selection pressure applied. These assays cannot dissect growth arrest, cell death, cell viability and target cell outgrowth, all of which occur as a consequence of released cytokines and direct tumor/T-cell interactions, hence, significantly impact the interpretation of small- and large-scale screens in co-culture models. These biases are particularly relevant when comparing cell lines or genotypes with different growth kinetics. Here, we use a dual reporter system and adopt pharmaco-mathematical principles to dissect these biases to create a growth-rate corrected co-culture metric (GRC), which normalizes T-cell mediated antiproliferative and cytotoxic effects in tumor target cells. We illustrate the value of this metric by comparing fast and slow-growing tumor cells in autologous patient-derived melanoma/T-cell co-culture pairs. At tumor-T-cell ratios resulting in a half maximal relative reduction of target cell counts compared to controls, the fast-proliferating cell line had not changed in absolute cell counts since baseline while the slow-growing cell line showed a 50% reduction. This indicates that in one instance (fast growing cell line) growth-retardation occurred due to a balance of immune editing and target cell proliferation, while in the other (slow-growing cell line) T-cell mediated killing resulted in net loss of tumor cells. Using GRC, these differences can be readily distinguished, and comparable selection conditions can be selected. This has important implications for large-scale screens with perturbations within cancer cells: drop-outs from a screen in the fast-growing cell line would not only contain perturbations that increase specific sensitivity to T-cell mediated cytotoxicity, but also perturbations causing a general loss in proliferation/survival fitness. In addition, some of the discrepancy in recently published screens may be explained by differences in models with varying proliferation rates and therefore selection conditions. In summary, the GRC metric enhances the interpretation of co-culture experiments and could contribute to improved selection of targets from large-scale screens investigating resistance to T-cell mediated toxicity for drug development. Citation Format: Johannes Christian Melms, Clarence Yapp, Caitlin Elizabeth Mills, Peter Sorger, Benjamin Izar. A growth rate corrected metric for cytotoxic co-culture systems [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 2168.

BACKGROUND: Advances in genomic profiling, together with a better understanding of cancer genetic drivers have generated opportunities for precision medicine trials in newly diagnosed glioblastoma (GBM). ALLELE is a consortium to create independent infrastructure for prospective genomics and functional diagnostics to support biomarker-driven trials in GBM. METHODS: Multi-center prospective study of molecular profiling in newly diagnosed GBM. Clinical (CLIA) tumor/normal whole exome sequencing (WES) and genome-wide copy number array (CNA) were performed. Primary objective: to evaluate the feasibility of genotyping tumors with a turnaround time allowing prospective data use in prospective trials. Secondary objectives included developing infrastructure for novel functional biomarker assays and investigating the clinical yield of tumor/normal WES and CNA in GBM. RESULTS: As of 5/1/18, 65 patients with GBM enrolled among 7 sites. Median age was 59. WES and CMA were completed in 60 patients, with a median time between tissue submission and reporting of 22 days (range 15–35). 33 patients were enrolled in INSIGhT, a randomized platform adaptive trial comparing standard of care versus adjuvant CC-115, neratinib or abemaciclib in MGMT unmethylated newly diagnosed GBM (NCT02977780). In each arm, pre-defined biomarkers (EGFR, PI3K and CDK) will be evaluated for their ability to predict outcome. Potentially actionable findings were identified in 30 patients, and included EGFR amplification, mutations of BRAF, FGFR1, IDH1/2 or FGFR3 or MET fusion. Four tumors were reclassified based on genomics. Functional biomarker assays were developed for real-time evaluation of pharmacodynamic responses in ex vivo models. Updated results of exploratory biomarker analyses will be presented at the conference. CONCLUSIONS: Real-time WES, copy number arrays and functional diagnostics are feasible in newly diagnosed glioblastoma, and can support a variety of biomarker-driven trials. Genomic analyses conducted in a prospective manner can inform subsequent clinical trial analysis aiming at matching outcome with tumor genotyping.

Summary Cyclin-dependent kinase 7 (CDK7) regulates both cell cycle and transcription, but its precise role remains elusive. We previously described THZ1, a CDK7 inhibitor, which dramatically inhibits superenhancer-associated gene expression. However, potent CDK12/13 off-target activity obscured CDK7s contribution to this phenotype. Here, we describe the discovery of a highly selective covalent CDK7 inhibitor. YKL-5-124 causes arrest at the G1/S transition and inhibition of E2F-driven gene expression; these effects are rescued by a CDK7 mutant unable to covalently engage YKL-5-124, demonstrating on-target specificity. Unlike THZ1, treatment with YKL-5-124 resulted in no change to RNA polymerase II C-terminal domain phosphorylation; however, inhibition could be reconstituted by combining YKL-5-124 and THZ531, a selective CDK12/13 inhibitor, revealing potential redundancies in CDK control of gene transcription. These findings highlight the importance of CDK7/12/13 polypharmacology for anti-cancer activity of THZ1 and posit that selective inhibition of CDK7 may be useful for treatment of cancers marked by E2F misregulation.

SUMMARY Evidence that some high-impact biomedical results cannot be repeated has stimulated interest in practices that generate findable, accessible, interoperable, and reusable (FAIR) data. Multiple papers have identified specific examples of irreproducibility, but practical ways to make data more reproducible have not been widely studied. Here, five research centers in the NIH LINCS Program Consortium investigate the reproducibility of a prototypical perturbational assay: quantifying the responsiveness of cultured cells to anti-cancer drugs. Such assays are important for drug development, studying cellular networks, and patient stratification. While many experimental and computational factors impact intra- and inter-center reproducibility, the factors most difficult to identify and control are those with a strong dependency on biological context. These factors often vary in magnitude with the drug being analyzed and with growth conditions. We provide ways to identify such context-sensitive factors, thereby improving both the theory and practice of reproducible cell-based assays., Graphical Abstract, In Brief Factors that impact the reproducibility of experimental data are poorly understood. Five NIH-LINCS centers performed the same set of drug-response measurements and compared results. Technical and biological variables that impact precision and reproducibility and are also sensitive to biological context were the most problematic.

The target profiles of many drugs are established early in their development and are not systematically revisited at the time of approval, raising the question whether human therapeutics with the same nominal targets but different chemical structures are functionally equivalent. In this paper we use five distinct phenotypic and biochemical assays to compare approved inhibitors of the cyclindependent kinases CDK4/6 (palbociclib, ribociclib, and abemaciclib) that are promising therapies for hormone-receptor positive breast cancer. We find that transcriptional, proteomic and phenotypic changes induced by the three drugs differ significantly and that abemaciclib has activates overlapping those of the older generation CDK inhibitor alvocidib. Abemaciclib’s activities arise from inhibition of kinases other than CDK4/6 including CDK2/Cyclin A/E and CDK1/Cyclin B. Our data suggest the potential for differential use in the clinic and argue that a multi-faceted experimental and computational approach is necessary to obtain a reliable picture of kinase inhibitor target spectrum.

Mammography is used to screen a large fraction of the population for breast cancer, and mammography quality X rays are speculated to be more damaging than the higher energy X rays used for other diagnostic procedures. The radiation dose delivered to breast cells as a result of these screening exposures may be a concern. The purpose of this current study was to determine the relative biological effectiveness (RBE) of low-energy mammography X rays for radiation-induced DNA double-strand breaks evaluated using a highly sensitive automated 53BP1 assay. Automation of the 53BP1 assay enabled the quantification and analysis of meaningful image-based features, including foci counting, within the cell nuclei. Nontumorigenic, human breast epithelial MCF-10A cells were irradiated in the low-dose range with approximately 3-30 mGy of 29 kVp mammography X rays or (137)Cs (662 keV) gamma rays. The induction and resolution of the 53BP1 foci did not differ significantly between exposures to (137)Cs gamma rays and 29 kVp X rays. The RBE was calculated to be 1.1 with a standard deviation of 0.2 for the initial number of radiation-induced double-strand breaks. The radiation dose from a single mammogram did not yield a significant change in the number of detectable foci. However, analysis of additional features revealed subtle differences in the distribution of 53BP1 throughout the nuclei after exposure to the different radiation qualities. A single mammogram was sufficient to alter the distribution of 53BP1 within the nuclear area, but not into discrete foci, while a dose-matched gamma exposure was not sufficient to alter the distribution of 53BP1. Our results indicate that exposure to clinically relevant doses of low-energy mammography quality X rays does not induce more DNA double-strand breaks than exposure to higher energy photons.

SUMMARYThe target profiles of many drugs are established early in their development and are not systematically revisited at the time of FDA approval. Thus, it is often unclear whether therapeutics with the same nominal targets but different chemical structures are functionally equivalent. In this paper we use five different phenotypic and biochemical assays to compare approved inhibitors of cyclin-dependent kinases 4/6 – collectively regarded as breakthroughs in the treatment of hormone receptor-positive breast cancer. We find that transcriptional, proteomic and phenotypic changes induced by palbociclib, ribociclib, and abemaciclib differ significantly; abemaciclib in particular has advantageous activities partially overlapping those of alvocidib, an older polyselective CDK inhibitor. In cells and mice, abemaciclib inhibits kinases other than CDK4/6 including CDK2/Cyclin A/E – implicated in resistance to CDK4/6 inhibition – and CDK1/Cyclin B. The multi-faceted experimental and computational approaches described here therefore uncover under-appreciated differences in CDK4/6 inhibitor activities with potential importance in treating human patients.

The G1/S cell cycle checkpoint is frequently dysregulated in cancer, leaving cancer cells reliant on a functional G2/M checkpoint to prevent excessive DNA damage. Wee1 regulates the G2/M checkpoint by phosphorylating CDK1 at Tyr15 to prevent mitotic entry. Previous drug development efforts targeting Wee1 resulted in the clinical-grade inhibitor, AZD1775. However, AZD1775 is burdened by dose-limiting adverse events, and has off-target PLK1 activity. In an attempt to overcome these limitations, we developed Wee1 degraders by conjugating AZD1775 to the cereblon (CRBN)-binding ligand, pomalidomide. The resulting lead compound, ZNL-02-096, degrades Wee1 while sparing PLK1, induces G2/M accumulation at 10-fold lower doses than AZD1775, and synergizes with Olaparib in ovarian cancer cells. We demonstrate that ZNL-02-096 has CRBN-dependent pharmacology that is distinct from AZD1775, which justifies further evaluation of selective Wee1 degraders.

Frequent mutation of PI3K/AKT/mTOR signaling pathway genes in human cancers has stimulated large investments in targeted drugs but clinical successes are rare. As a result, many cancers with high PI3K pathway activity, such as triple-negative breast cancer (TNBC), are treated primarily with chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors, we find that one drug, Torin2, is unusually effective because it inhibits both mTOR and other PI3K-like kinases (PIKKs). In contrast to mTOR-selective inhibitors, Torin2 exploits dependencies on several kinases for S-phase progression and cell-cycle checkpoints, thereby causing accumulation of single-stranded DNA and death by replication catastrophe or mitotic failure. Thus, Torin2 and its chemical analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors.

Frequent mutation of genes in the PI3K/AKT/mTOR signaling pathway in human cancers has stimulated large investments in over 40 small molecule therapeutic drugs but most have low efficacy in patients. As a result, cancers with high PI3K pathway activity such as triple-negative breast cancer (TNBC) are still treated primarily with conventional chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors with varying degrees of polyselectivity, we find that the preclinical compound Torin2 and related analogs are unusually effective because they inhibit both mTORC1/2 and structurally related PI3K-like kinases (PIKKs). The activity of Torin2 in TNBC cells is most closely mimicked by combining selective inhibitors of mTORC1/2 and Chk1, a kinase that acts downstream of the PIKKs ATR and DNA-PK in the signaling response to replication stress. Torin2 is more cytotoxic in TNBC/basal-like cell lines (N=19) than clinical grade mTORC1/2 inhibitors such as sapanisertib, but produces no cytotoxicity in non-transformed cells with basal-like gene expression (N=2). The high activity of Torin2 in TNBC is associated with greater activation of caspase-dependent apoptosis and stronger inhibition of cell proliferation than conventional PI3K/AKT/mTOR inhibitors, which do not inhibit PIKKs. Pulse-labeling studies with thymidine analogs and live-cell imaging of cells expressing fluorescent cell cycle reporters show that unlike other PI3K pathway drugs, Torin2 blocks progression of S phase, thereby causing accumulation of single-stranded DNA, DNA damage and death by replication catastrophe or mitotic failure. These phenotypes appear to result from co-targeting distinct pathways required during S phase in TNBC cells: inhibiting mTOR perturbs de novo biosynthesis and salvage of pyrimidines and purines, and inhibiting PIKKs causes high levels of replication stress and DNA damage. Moreover, computational models developed to better understand the complex cell cycle mechanism of action of Torin2 demonstrate the importance of co-targeting S-phase vulnerabilities for cytotoxicity. Thus, Torin2 and related analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC and possibly to other cancer cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors. Citation Format: Sameer S. Chopra, Annie Jenney, Adam Palmer, Mario Niepel, Mirra Chung, Caitlin Mills, Sindhu Carmen Sivakumaren, Qingsong Liu, Jia-Yun Chen, Clarence Yapp, Nathanael S. Gray, Peter K. Sorger. Torin2 and related analogs exploit replicative and checkpoint vulnerabilities to induce death of triple-negative breast cancer cells [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 B103. doi:10.1158/1535-7163.TARG-19-B103

Oncogene-targeted therapies are initially effective in most patients with BRAF-mutated melanoma. However, patients frequently develop resistance during therapy. At the time of therapy resistance, they are also less likely to respond to immune checkpoint inhibitors (ICI) with response rates ranging from 0-12%, compared to 40-60% when used in the first line. This may be in part be due to rapid tumor growth that outpaces pharmacodynamics of ICI. New drugs that directly control tumor growth and effectively enhance ICI activity are therefore needed. We report that the small molecule CX-6258 has potent activity against both RAF/MEK inhibitor (RMS) and resistant (RMR) BRAF-mutant melanoma cell lines. CX-6258 is annotated as a pan-PIM kinase inhibitor, with an EC50 corrected for growth rate (GR50) of ~200 nM; in contrast, other PIM inhibitors had no in vitro activity, suggesting that PIMs may not be the true target in melanoma - we therefore sought to systematically study the targets of this compound. Using an unbiased kinase profiling approach, we identified Haspin Kinase (HASPIN) as the key target of CX-6258 with an IC50~10nM. Haspin kinase (Haspin) is a poorly understood mitotic kinase, whose only well-defined function is phosphorylation of Histon H3 at Thr3 (pH3T3). Treatment with CX-6258 resulted in reduced proliferation, reduced phosphorylation of pH3T3, frequent formation of micronuclei (MN), recruitment of cGAS and activation of the cGAS-STING-pathway. By using CRISPR/CAS9 knock outs of PIM kinase we show that these targets are not required for CX-6258 function. Furthermore, siRNA mediated knock-down of HASPIN phenocopied CX-6258. In murine models, CX-6258 induces a potent cGAS dependent type-I-interferon response in tumor cells, increases IFNγ-producing CD8+ T-cells, reduces Tregs, and enhances responses to ICIs in vivo. HASPIN is more strongly expressed in malignant compared to healthy tissue and its inhibition by CX-6258 has minimal toxicity in ex vivo expanded human tumor-infiltrating-lymphocytes (TILs) and in vitro differentiated human neurons, suggesting a potential therapeutic index for anti-cancer therapy. CX-6258 also shows activity in several Ewing sarcoma and multiple myeloma cell lines with. In summary, HASPIN inhibition may overcome drug resistance in melanoma, synergize with ICIs and target a vulnerability in different cancer lineages. Citation Format: Johannes C. Melms, Sreeram Vallabhaneni, Caitlin E. Mills, Kai Wucherpfennig, Peter K. Sorger, Benjamin Izar. Inhibition of Haspin kinase to promote cell-intrinsic and extrinsic anticancer therapy [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 B132. doi:10.1158/1535-7163.TARG-19-B132

Intrinsic or acquired resistance to targeted therapies, such as RAF/MEK inhibitors in melanoma, represents a significant clinical challenge. We performed a small molecule screen in isogenic sensitive (S) and RAF/MEK-inhibitor-resistant (RMR) melanoma cell lines and identified a nominal PIM kinase inhibitor (Comp-1) with high activity against A375-S and A375-RMR, with an EC50of ~100 nM. While the phosphorylation of some PIM targets, including 4EBP1 and MYC, were significantly inhibited in a dose-dependent fashion, phosphorylation of BAD was not reduced as one would expect. To determine the on-target activity of this compound, we generated A375 cell lines overexpressing WT PIM isoforms 1-3 or their kinase-dead (KD) forms, followed by treatment with PIMi-1. We found only modest shifts in EC50and hypothesized that the beneficial effects of this compounds were due to inhibition of an alternative target. A screen of Comp-1, along with structurally related compounds, against 468 human kinases revealed exquisite affinity for Haspin kinase (HK). We determined the IC50of Comp-1 against HK to be 14.9 nM. The only described role of HK is phosphorylation of Histone H3 at Thr 3 (H3T3) and thereby regulation of mitotic progression. Quantitative single-cell immunofluorescence (IF) analysis of Comp-1 treated A375 confirmed complete abrogation of p-H3T3, indicating on-target activity in vitro. To understand the temporal effects of HK inhibition with Comp-1, we performed live-cell imaging and single-cell analysis of A375 transduced with a mitotic phase-specific reporter. Cells experienced mitotic stress or mitotic slippage resulting in DNA-damage and an increase gamma-H2AX foci per nuclei. In line with these observation, we observed increased phosphorylation p53 and CHECK2. Furthermore, a portion of cells developed micronuclei in a dose-dependent fashion providing a substrate for the cytosolic DNA-sensing machinery, including the cGAS-STING pathway, and rationale for combining Comp-1 with immunotherapies. Ongoing experiments in tumor-bearing BALB/c mice indicate safety and potentially enhanced efficacy of Comp-1 when combined with either anti-CTLA-4 or anti-PD-1 therapy. Ex vivotreatment of patient-derived tumor-infiltrating lymphocytes (TILs) with Comp-1 revealed no toxic effects. A pan-cancer RNA-seq analysis revealed significant upregulation of HK in 34 of 37 tumor types supporting its role as a potential therapeutic target. Overall, our work identified a highly selective Haspin Kinase inhibitor with activity against RAF/MEK inhibitor resistant melanoma, insights into the underlying mechanisms, and potential in vivo benefit when combined with immune checkpoint inhibitors. Citation Format: Johannes C. Melms, Sreeram Vallabhaneni, Caitlin E. Mills, Peter K. Sorger, Kai Wucherpfennig, Benjamin Izar. A highly selective dual Haspin-kinase and PIM-inhibitor overcomes RAF/MEK-inhibitor resistance in melanoma [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 2068.

FDA approval of multiple drugs with different chemical structures against the same protein target raises the question whether such drugs have sufficiently similar mechanisms of action to be considered functionally equivalent. We compare three recently approved inhibitors of the cyclin-dependent kinases CDK4/6 - palbociclib, ribociclib, and abemaciclib - that have become highly promising therapies for the treatment of breast cancer and potentially other solid tumors. All three drugs have the same nominal targets but differ in selectivity. In humans, abemaciclib uniquely appears to exhibit single-agent activity and has a distinct toxicity profile. We systematically profile targets and activities of these CDK4/6 inhibitors using biochemical assays, mRNA profiling, mass spectrometry-based phospho-proteomics, GR-based dose-response assays, and mouse xenografts. We find that the three drugs differ at a cellular level and that abemaciclib has targets and activities not shared by palbociclib or ribociclib including: induction of cell death (even in pRb-deficient cells), arrest in the G2 phase of the cell cycle, reduced drug adaptation, and unique transcriptional effects in vitro and in vivo. These activities appear to arise from inhibition of CDKs other than CDK4/6 including CDK2/Cyclin A/E and CDK1/Cyclin B. We propose that inhibition of these kinases by abemaciclib target known mechanisms of resistance to CDK4/6 inhibition and thus elicit a response in cell lines that are resistant to palbociclib or ribociclib. Citation Format: Caitlin E. Mills, Marc A. Hafner, Kartik Subramanian, Chen Chen, Mirra Chung, Sarah A. Boswell, Robert A. Everley, Changchang Liu, Charlotte S. Walmsley, Dejan Juric, Peter K. Sorger. Multi-omics profiling establishes the polypharmacology of FDA Approved CDK4/6 inhibitors and its impact on drug response [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 4432.

The recent introduction of small-molecule inhibitors of cyclin-dependent kinases (CDK) 4/6 to the clinic has improved the treatment of hormone receptor-positive breast cancer, and shown promise in other malignancies. The three clinically used CDK4/6 inhibitors, palbociclib, ribociclib, and abemaciclib, are reported to be broadly similar, although recent data suggest that abemaciclib has distinct single-agent activity in patients and a unique toxicity profile. In vitro profiling of the CDK4/6 inhibitors has shown abemaciclib to be more potent on target, but less selective than either palbociclib or ribociclib. We sought to systematically investigate the functional consequences of abemaciclib’s polypharmacology. We used molecular and phenotypic profiling by mRNA sequencing, mass spectrometry-based proteomics, and GR-based dose-response assays to characterize the mechanisms of action of the three CDK4/6 inhibitors. Inhibition of CDKs other than CDK4/6 by abemaciclib resulted in an arrest in the G2 phase of the cell cycle, a prolonged inhibition of growth, and cytotoxicity, even in retinoblastoma protein (pRb)-deficient cells. Abemaciclib elicited unique molecular responses at clinically achievable concentrations that are likely to be therapeutically advantageous and prevented cross-resistance with ribociclib and palbociclib. More generally, we propose that multi-omic approaches are required to fully elucidate the spectrum of targets relevant to drug mechanisms of action in cells, and we expect such understanding to assist in stratifying patient populations and in ordering sequential therapies when resistance arises. Citation Format: Caitlin E. Mills, Marc Hafner, Kartik Subramanian, Chen Chen, Sarah A. Boswell, Robert A. Everley, Dejan Juric, Peter K. Sorger. Omics profiling of CDK4/6 inhibitors reveals functionally important secondary targets of abemaciclib [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B60.