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In vitro cell line IC50 values and mutational status.

In vitro response to co-inhibition of MAPK and mTOR pathways in BRAFV600E mutated AM-38 glioma cells.

Purpose: Alteration of the BRAF/MEK/MAPK pathway is the hallmark of pediatric low-grade gliomas (PLGGs), and mTOR activation has been documented in the majority of these tumors. We investigated combinations of MEK1/2, BRAFV600E and mTOR inhibitors in gliomas carrying specific genetic alterations of the MAPK pathway.Experimental Design: We used human glioma lines containing BRAFV600E (adult high-grade: AM-38, DBTRG, PLGG: BT40), or wild-type BRAF (pediatric high-grade: SF188, SF9427, SF8628) and isogenic systems of KIAA1549:BRAF-expressing NIH/3T3 cells and BRAFV600E-expressing murine brain cells. Signaling inhibitors included everolimus (mTOR), PLX4720 (BRAFV600E), and AZD6244 (MEK1/2). Proliferation was determined using ATP-based assays. In vivo inhibitor activities were assessed in the BT40 PLGG xenograft model.Results: In BRAFV600E cells, the three possible doublet combinations of AZD6244, everolimus, and PLX4720 exhibited significantly greater effects on cell viability. In BRAFWT cells, everolimus + AZD6244 was superior compared with respective monotherapies. Similar results were found using isogenic murine cells. In KIAA1549:BRAF cells, MEK1/2 inhibition reduced cell viability and S-phase content, effects that were modestly augmented by mTOR inhibition. In vivo experiments in the BRAFV600E pediatric xenograft model BT40 showed the greatest survival advantage in mice treated with AZD6244 + PLX4720 (P < 0.01).Conclusions: In BRAFV600E tumors, combination of AZD6244 + PLX4720 is superior to monotherapy and to other combinatorial approaches. In BRAFWT pediatric gliomas, everolimus + AZD6244 is superior to either agent alone. KIAA1549:BRAF-expressing tumors display marked sensitivity to MEK1/2 inhibition. Application of these results to PLGG treatment must be exercised with caution because the dearth of PLGG models necessitated only a single patient-derived PLGG (BT40) in this study. Clin Cancer Res; 22(21); 5312–21. ©2016 AACR.

Combinatorial index and median-effect plots for BRAFV600E mutated cell lines.

Combinatorial index and median-effect plots for BRAFWT cell lines.

Kinase inhibitors are used widely to treat various cancers, but adaptive reprogramming of kinase cascades and activation of feedback loop mechanisms often contribute to therapeutic resistance. Determining comprehensive, accurate maps of kinase circuits may therefore help elucidate mechanisms of response and resistance to kinase inhibitor therapies. In this study, we identified and validated phosphorylatable target sites across human cell and tissue types to generate PhosphoAtlas, a map of 1,733 functionally interconnected proteins comprising the human phospho-reactome. A systematic curation approach was used to distill protein phosphorylation data cross-referenced from 38 public resources. We demonstrated how a catalog of 2,617 stringently verified heptameric peptide regions at the catalytic interface of kinases and substrates could expose mutations that recurrently perturb specific phospho-hubs. In silico mapping of 2,896 nonsynonymous tumor variants identified from thousands of tumor tissues also revealed that normal and aberrant catalytic interactions co-occur frequently, showing how tumors systematically hijack, as well as spare, particular subnetworks. Overall, our work provides an important new resource for interrogating the human tumor kinome to strategically identify therapeutically actionable kinase networks that drive tumorigenesis. Cancer Res; 76(7); 1733–45. ©2016 AACR.

Legends for Supplementary Figures S1-S17.

Phospho-reactome curation workflow (S1); Proteins catalogued in PhosphoAtlas correspond to a unique subset of Protein-Protein Interactions, defined by their active, proven function in phospho-catalytic events (S2); Comparative connectivity between kinases, substrates and HPS's (S3); Most highly connected kinases and substrates (S4); Comparison between the number of enzyme - substrate pairs and the number of kinase - HPS - substrate connections across kinase subfamilies (S5); Disease ontology profile of AKT phospho-circuits (S6); Connectivity of AKT proteins to their upstream and downstream phospho-protein circuits (S7); Catalytic circuitry of AKT1 kinase: an expandable network of enzymatic interaction derived from mutation-prone phospho-target sites (S8); Identification of substrate proteins that are most affected by cancer-mutated peptides, and substrates that are not subject to alterations of their phospho-target sites (S9); Distribution and classification of SNP-altering target sites (S10); Heptameric target variants affecting phosphorylation sites mapped to COSMIC-curated tissue pathologies (S11); Identification of kinases impacted by cancer-mutations that alter their phosphorylatable target sites (S12); Map of the tumor kinome (S13, S14); Examples of kinase and substrate sub-networks, and the differential impact of tumor mutations on their phospho-signaling circuits (S15, S16, S17).

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Background Information regarding response to past treatments may provide clues concerning the classes of drugs most or least likely to work for a particular metastatic or neoadjuvant early stage breast cancer patient. However, currently there is no systematized knowledge base that would support clinical treatment decision-making that takes response history into account. Methods To model history-dependent response data we leveraged a published in vitro breast cancer viability dataset (84 cell lines, 90 therapeutic compounds) to calculate the odds ratios (log (OR)) of responding to each drug given knowledge of (intrinsic/prior) response to all other agents. This OR matrix assumes (1) response is based on intrinsic rather than acquired characteristics, and (2) intrinsic sensitivity remains unchanged at the time of the next decision point. Fisher’s exact test is used to identify predictive pairs and groups of agents (BH p Results Of the 90 compounds, 57 have sensitivity profiles significantly associated with those of at least one other agent, mostly targeted drugs. Nearly all associations are positive, with (intrinsic/prior) sensitivity to one agent predicting sensitivity to others in the same or a related class (OR > 1). In vitro conditional response patterns clustered compounds into five predictive classes: (1) DNA damaging agents, (2) Aurora A kinase and cell cycle checkpoint inhibitors; (3) microtubule poisons; (4) HER2/EGFR inhibitors; and (5) PIK3C catalytic subunit inhibitors. The apriori algorithm implementation made further predictions including a directional association between resistance to HER2 inhibition and sensitivity to proteasome inhibitors. Conclusions Investigating drug sensitivity conditioned on observed sensitivity or resistance to prior drugs may be pivotal in informing clinicians deciding on the next line of breast cancer treatments for patients who have progressed on their current treatment. This study supports a strategy of treating patients with different agents in the same class where an associated sensitivity was observed, likely after one or more intervening treatments.

Target identification is a critical pillar within the drug discovery process that involves deconvoluting the protein target of a pharmacologically active small molecule ligand. While photoaffinity labeling strategies have become the benchmark for target deconvolution of small molecules owing to their reliance on external activation to induce covalent protein capture, the process of target identification remains one of the most technically challenging aspects of early drug discovery. Thus, there is a strong demand for new technologies that allow for controlled activation of chemical probes to covalently label their protein target. Here, we introduce an electroaffinity labeling (ECAL) platform which leverages the use of a small, redox-active diazetidinone (DZE) functional group to enable chemoproteomic-based target identification of pharmacophores within live cell environments.

State-of-the art photoactivation strategies in chemical biology provide spatiotemporal control and visualization of biological processes. However, using high energy light (λ < 500 nm) for substrate or photocatalyst sensitization can lead to background activation of photoactive small molecule probes and reduce its efficacy in complex biological environments. Here we describe the development of targeted aryl azide activation via deep red light (λ = 660 nm) photoredox catalysis and its use in photocatalyzed proximity labeling. We demonstrate that aryl azides are converted to triplet nitrenes via a novel redox-centric mechanism and show that its spatially localized-formation requires both red light and a photocatalyst-targeting modality. This technology was applied in different colon cancer cell systems for targeted protein environment labeling of epithelial cell adhesion molecule (EpCAM). We identified a small subset of proteins with previously known and unknown association to EpCAM, including CDH3, a clinically relevant protein that shares high tumor selective expression with EpCAM.

State-of-the art photoactivation strategies in chemical biology provide spatiotemporal control and visualization of biological processes. However, using high energy light (λ < 500 nm) for substrate or photocatalyst sensitization can lead to background activation of photoactive small molecule probes and reduce its efficacy in complex biological environments. Here we describe the development of targeted aryl azide activation via deep red light (λ = 660 nm) photoredox catalysis and its use in photocatalyzed proximity labeling. We demonstrate that aryl azides are converted to triplet nitrenes via a novel redox-centric mechanism and show that its spatially localized-formation requires both red light and a photocatalyst-targeting modality. This technology was applied in different colon cancer cell systems for targeted protein environment labeling of epithelial cell adhesion molecule (EpCAM). We identified a small subset of proteins with previously known and unknown association to EpCAM, including CDH3, a clinically relevant protein that shares high tumor selective expression with EpCAM.

Elicitation of tumor cell killing by CD8+ T cells is an effective therapeutic approach for cancer. In addition to using immune checkpoint blockade to reinvigorate existing but unresponsive tumor-specific T cells, alternative therapeutic approaches have been developed, including stimulation of polyclonal T cell cytolytic activity against tumors using bispecific T cell engager (BiTE) molecules that simultaneously engage the TCR complex and a tumor-associated Ag. BiTE molecules are efficacious against hematologic tumors and are currently being explored as an immunotherapy for solid tumors. To understand mechanisms regulating BiTE molecule­–mediated CD8+ T cell activity against solid tumors, we sought to define human CD8+ T cell populations that efficiently respond to BiTE molecule stimulation and identify factors regulating their cytolytic activity. We find that human CD45RA+CCR7− CD8+ T cells are highly responsive to BiTE molecule stimulation, are enriched in genes associated with cytolytic effector function, and express multiple unique inhibitory receptors, including leukocyte Ig-like receptor B1 (LILRB1). LILRB1 and programmed cell death protein 1 (PD1) were found to be expressed by distinct CD8+ T cell populations, suggesting different roles in regulating the antitumor response. Engaging LILRB1 with its ligand HLA-G on tumor cells significantly inhibited BiTE molecule–induced CD8+ T cell activation. Blockades of LILRB1 and PD1 induced greater CD8+ T cell activation than either treatment alone. Together, our data suggest that LILRB1 functions as a negative regulator of human CD8+ effector T cells and that blocking LILRB1 represents a unique strategy to enhance BiTE molecule therapeutic activity against solid tumors.

Background: Information regarding response to past treatments may provide clues concerning the classes of drugs most or least likely to work for a particular metastatic or neoadjuvant early stage breast cancer patient. However, currently there is no systematized knowledge base that would support clinical treatment decision-making that takes response history into account.Methods: To model history-dependent response data we leveraged a published in vitro breast cancer viability dataset (84 cell lines, 90 therapeutic compounds) to calculate the odds ratios (log(OR)) of responding to each drug given knowledge of (intrinsic/prior) response to all other agents. This OR matrix is assuming (1) response is based on intrinsic rather than acquired characteristics, and (2) intrinsic sensitivity remains unchanged at the time of the next decision point. Fisher’s exact test is used to identify predictive pairs and groups of agents (BH pResults: Of the 90 compounds, 57 have sensitivity profiles significantly associated with those of at least one other agent, mostly targeted drugs. Nearly all associations are positive, with (intrinsic/prior) sensitivity to one agent predicting sensitivity to others in the same or a related class (OR>1). In vitro conditional response patterns clustered compounds into five predictive classes: (1) DNA damaging agents, (2) Aurora A kinase and cell cycle checkpoint inhibitors; (3) microtubule poisons; (4) HER2/EGFR inhibitors; and (5) PIK3C catalytic subunit inhibitors. The apriori algorithm implementation made further predictions including a directional association between resistance to HER2 inhibition and sensitivity to proteasome inhibitors.Conclusions: Investigating drug sensitivity conditioned on observed sensitivity or resistance to prior drugs may be pivotal in informing clinicians deciding on the next line of breast cancer treatments for patients who have progressed on their current treatment. This study supports a strategy of treating patients with different agents in the same class where sensitivity was previously observed.

Purpose Few tools exist to predict clinical outcomes after radiopharmaceutical therapy. Our goal was to identify associations between blood-based biomarkers of radiation effect and clinical outcomes after 131 I-metaiodobenzylguanidine ( 131 I-MIBG) therapy in patients with neuroblastoma. Methods and Materials We conducted a prospective, single-center cohort study in children with advanced neuroblastoma treated with 131 I-MIBG as monotherapy or in combination with systemic putative radiation sensitizers. We collected serial peripheral blood samples after 131 I-MIBG infusions and quantified a panel of protein and messenger RNA markers. We plotted relative change from baseline to assess degree of modulation over time and then evaluated association of marker modulation with toxicity and response endpoints. Results The cohort included 40 patients (30 male/10 female; median age 7 years). We observed significant modulation of the majority of markers between baseline and hour 72 after 131 I-MIBG. Greater fold increase of plasma FLT3 ligand was associated with subsequent grade 4 neutropenia ( P =.039). Modulation of peripheral blood BCLXL and DDB2 was associated with grade 3+ nonhematologic toxicity ( P =.043 and .048, respectively). No markers were associated with tumor response. Greater plasma FLT3 ligand, BCLXL , and BCL2 modulation was observed in patients receiving 131 I-MIBG in combination with radiation sensitizers. Among 9 patients who received 2 courses, the degree of modulation in serum amylase was significantly lower after the second course ( P =.012). Conclusions Peripheral blood biomarkers relevant to radiation exposure are significantly modulated during the acute period after 131 I-MIBG. The degree of modulation of a subset of these markers is associated with toxicity and receipt of concomitant radiation sensitizers.

// Payal Jain 1,2,3 , Amanda Silva 1 , Harry J. Han 2 , Shih-Shan Lang 1,2 , Yuankun Zhu 1,3 , Katie Boucher 1,2,3 , Tiffany E. Smith 1,2,3 , Aesha Vakil 4 , Patrick Diviney 5 , Namrata Choudhari 1,2,3 , Pichai Raman 3,6,7 , Christine M. Busch 8 , Tim Delaney 1,2,3 , Xiaodong Yang 9 , Aleksandra K. Olow 10 , Sabine Mueller 9,11,12 , Daphne Haas-Kogan 13 , Elizabeth Fox 8 , Phillip B. Storm 1,2,3 , Adam C. Resnick 1,2,3,6,* and Angela J. Waanders 3,8,14,* 1 Division of Neurosurgery, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 2 Department of Neurosurgery, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA 3 Center for Data Driven Discovery in Biomedicine (D3b), The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 4 The Fred Hutchinson Cancer Research Center, Seattle, WA, USA 5 Division of Neurology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 6 Department of Biomedical and Health Informatics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 7 Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Philadelphia, PA, USA 8 Division of Oncology, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 9 Division of Neurology, University of California, San Francisco, CA, USA 10 Amgen, South San Francisco, CA, USA 11 Department of Neurosurgery, University of California, San Francisco, CA, USA 12 Department of Pediatrics, University of California, San Francisco, CA, USA 13 Department of Radiation Oncology, Harvard Medical School, Boston, MA, USA 14 Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA * These authors are co-senior authors and have contributed equally to this work Correspondence to: Angela J. Waanders, email: waandersA@email.chop.edu Keywords : pediatric low-grade glioma, BRAF-fusions, trametinib and everolimus, MAPK pathway, PI3K/mTOR pathway Received : March 03, 2017 Accepted : August 23, 2017 Published : September 15, 2017 Abstract Pediatric low-grade gliomas (PLGGs) are frequently associated with activating BRAF gene fusions, such as KIAA1549-BRAF, that aberrantly drive the mitogen activated protein kinase (MAPK) pathway. Although RAF inhibitors (RAFi) have been proven effective in BRAF-V600E mutant tumors, we have previously shown how the KIAA1549-BRAF fusion can be paradoxically activated by RAFi. While newer classes of RAFi, such as PLX8394, have now been shown to inhibit MAPK activation by KIAA1549-BRAF, we sought to identify alternative MAPK pathway targeting strategies using clinically relevant MEK inhibitors (MEKi), along with potential escape mechanisms of acquired resistance to single-agent MAPK pathway therapies. We demonstrate effectiveness of multiple MEKi against diverse BRAF-fusions with novel N-terminal partners, with trametinib being the most potent. However, resistance to MEKi or PLX8394 develops via increased RTK expression causing activation of PI3K/mTOR pathway in BRAF-fusion expressing resistant clones. To circumvent acquired resistance, we show potency of combinatorial targeting with trametinib and everolimus, an mTOR inhibitor (mTORi) against multiple BRAF-fusions. While single-agent mTORi and MEKi PLGG clinical trials are underway, our study provides preclinical rationales for using MEKi and mTORi combinatorial therapy to stave off or prevent emergent drug-resistance in BRAF-fusion driven PLGGs.

Elicitation of tumor cell killing by CD8

Background: Triple-negative breast cancer (TNBC) accounts for 10-15% of all breast cancer cases. A major area of innovation in TNBC is identifying potential treatment targets, especially in TNBC cells which survive chemotherapy. Our previous study showed that TNBC cells displayed deregulated kinase-dependent signaling cascades, and uniquely divergent phospho-circuits could be distinguished between TNBC vs non-TNBC cell lines [2014 SABCS abstract 1672, poster P2-05-09]. We further hypothesized that specific dysfunctional phospho-signaling network played a key role in the early adaptive changes in DNA damage response of TNBC cells exposed to DNA damaging chemotherapeutic agents. Methods: TNBC cell lines MDA-MB-231 and MDA-MB-436 were treated with 5-fluorouracil (5-Fu), carboplatin and doxorubicin at their respective half maximal inhibitory concentrations (ic50s). MiSeq gene sequencing of the untreated vs treated TNBC cells was performed to investigate whether exposure to chemotherapy agents for 3-day's duration would induce additional adaptive genetic mutation. Apoptosis and cell-cycle distribution of the untreated and treated TNBC cells were analyzed with flow cytometry. The functional phospho-signature of each TNBC cell sample was analyzed using a high throughput experimental platform that monitors the level of activity of myriad kinases at once. This technique used over 450 phospho-sensing probes, including over 150 controls in an aqueous-based assay to simultaneously and directly measure the phospho-catalytic activity of phosphorylating enzymes in cell lysates. The kinome activities of the untreated vs treated TNBC cell lines were compared respectively, and the most significantly deranged and functionally altered phospho-signaling cascades and their related kinases were identified as the early adaptive changes of the survived TNBC cells after the 3-day exposure to DNA damage chemotherapies. Results: Using the two TNBC cell lines treated with the three chemotherapies, we made 8 cell line samples, including 6 treated and 2 untreated as the control. MiSeq gene sequencing showed no significant additional adaptive genetic mutations in the treated TNBC cells after the 3-day short-term exposure to 5-Fu, carboplatin and doxorubicin. 36 phospho-signatures were generated and validated for repeatability and robustness. The kinase activity signature of each TNBC sample was analyzed and compared to each other using unsupervised hierarchical clustering. The phospho-sensing assay revealed that phospho-signaling cascades related to CHK1/2 and IKK kinases were differentially altered in the untreated vs treated TNBC cell lines, which, when respectively inhibited by AZD7762 and IKK16, successfully increased growth inhibition and cell death of TNBCs. Conclusions: We identified specific phospho-fingerprints of the early adaption of TNBC cell lines and combinatorial targeted therapies that improve treatment outcome. Our next goal is to identify specific phosphorylation cascades in a broader range of cell lines and tumor tissues, to explore the actionable, kinase-dependent mechanisms critical to the DNA damage-induced adaptive reprogramming of TNBCs and early changes driving drug-resistance. Citation Format: Pan B, Olow A, Sun Q, Mori M, Lee PRE, Hartog M, Wang C, Wolf D, Yau C, van 't Veer L, Coppé J-P. Functional detection and inhibition of the targetable oncogenic kinome of chemotherapy-treated triple-negative breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-08-03.

Few tools exist to predict clinical outcomes after radiopharmaceutical therapy. Our goal was to identify associations between blood-based biomarkers of radiation effect and clinical outcomes afterWe conducted a prospective, single-center cohort study in children with advanced neuroblastoma treated withThe cohort included 40 patients (30 male/10 female; median age 7 years). We observed significant modulation of the majority of markers between baseline and hour 72 afterPeripheral blood biomarkers relevant to radiation exposure are significantly modulated during the acute period after

Background A substantial proportion of solid tumors carry the BRAF V600E mutation, which causes activation of the MEK/MAPK pathway and is a poor prognostic indicator. Patients with locally advanced human cancers are often treated with external beam radiation therapy. Given the association of Raf overactivation with radioresistance, we hypothesized that, in BRAF V600E-mutated carcinomas, there would be combinatorial activity between radiation and PLX4720, a specific BRAF V600E-inhibitor. Methods Two BRAF V600E-mutated cancer cell lines and one BRAF-V600E wildtype (WT) cancer cell line were obtained. We performed cell viability assays and clonogenic assays using combinations of radiation and PLX4720. We assessed MEK and MAPK phosphorylation at different PLX4720 concentrations with western blotting, and cell cycle progression was evaluated by flow cytometry. Results Our results show combinatorial, additive activity between radiation and PLX4720 in BRAF V600E-mutated cell lines, but not in the BRAF WT line. In BRAF V600E-mutated cells, there was a PLX4720 concentration-dependent decrease in MEK and MAPK phosphorylation. In cells with BRAF V600E mutations, PLX4720 caused cell cycle arrest at G1, and, when combined with radiation, caused a combined G1 and G2 cell cycle arrest; this pattern of cell cycle effects was not seen in the BRAF WT cell line. Conclusions These data suggest additive, combinatorial activity between radiation and PLX4720 in cancers carrying BRAF V600E mutations. Our data has potential for translation into the multimodality treatment of BRAF V600E-mutated cancers.

Purpose: Alteration of the BRAF/MEK/MAPK pathway is the hallmark of pediatric low-grade gliomas (PLGGs), and mTOR activation has been documented in the majority of these tumors. We investigated combinations of MEK1/2, BRAFV600E and mTOR inhibitors in gliomas carrying specific genetic alterations of the MAPK pathway. Experimental Design: We used human glioma lines containing BRAFV600E (adult high-grade: AM-38, DBTRG, PLGG: BT40), or wild-type BRAF (pediatric high-grade: SF188, SF9427, SF8628) and isogenic systems of KIAA1549:BRAF-expressing NIH/3T3 cells and BRAFV600E-expressing murine brain cells. Signaling inhibitors included everolimus (mTOR), PLX4720 (BRAFV600E), and AZD6244 (MEK1/2). Proliferation was determined using ATP-based assays. In vivo inhibitor activities were assessed in the BT40 PLGG xenograft model. Results: In BRAFV600E cells, the three possible doublet combinations of AZD6244, everolimus, and PLX4720 exhibited significantly greater effects on cell viability. In BRAFWT cells, everolimus + AZD6244 was superior compared with respective monotherapies. Similar results were found using isogenic murine cells. In KIAA1549:BRAF cells, MEK1/2 inhibition reduced cell viability and S-phase content, effects that were modestly augmented by mTOR inhibition. In vivo experiments in the BRAFV600E pediatric xenograft model BT40 showed the greatest survival advantage in mice treated with AZD6244 + PLX4720 (P < 0.01). Conclusions: In BRAFV600E tumors, combination of AZD6244 + PLX4720 is superior to monotherapy and to other combinatorial approaches. In BRAFWT pediatric gliomas, everolimus + AZD6244 is superior to either agent alone. KIAA1549:BRAF-expressing tumors display marked sensitivity to MEK1/2 inhibition. Application of these results to PLGG treatment must be exercised with caution because the dearth of PLGG models necessitated only a single patient-derived PLGG (BT40) in this study. Clin Cancer Res; 22(21); 5312–21. ©2016 AACR.

Kinase inhibitors are used widely to treat various cancers, but adaptive reprogramming of kinase cascades and activation of feedback loop mechanisms often contribute to therapeutic resistance. Determining comprehensive, accurate maps of kinase circuits may therefore help elucidate mechanisms of response and resistance to kinase inhibitor therapies. In this study, we identified and validated phosphorylatable target sites across human cell and tissue types to generate PhosphoAtlas, a map of 1,733 functionally interconnected proteins comprising the human phospho-reactome. A systematic curation approach was used to distill protein phosphorylation data cross-referenced from 38 public resources. We demonstrated how a catalog of 2,617 stringently verified heptameric peptide regions at the catalytic interface of kinases and substrates could expose mutations that recurrently perturb specific phospho-hubs. In silico mapping of 2,896 nonsynonymous tumor variants identified from thousands of tumor tissues also revealed that normal and aberrant catalytic interactions co-occur frequently, showing how tumors systematically hijack, as well as spare, particular subnetworks. Overall, our work provides an important new resource for interrogating the human tumor kinome to strategically identify therapeutically actionable kinase networks that drive tumorigenesis. Cancer Res; 76(7); 1733–45. ©2016 AACR.

Triple-negative breast cancer (TNBC) accounts for approximately 15% of all breast cancer cases, with over 35,000 newly diagnosed women per year in the USA. TNBC patients are at high risk of recurrence, and neoadjuvant standard chemotherapy leads to pathologic complete response in only about 30% of patients. No targeted therapy has yet been conclusively established to improve outcome. The management of TNBC will significantly improve once mechanisms responsible for TNBC resistance to chemotherapies are identified. Here, we applied a new functional proteomic strategy to reveal which (dys)regulated phospho-signaling circuits are the effective dependencies of chemotherapy-resistant TNBC cells. The high-throughput kinase activity-mapping (HT-KAM) assay is our new screening technology to assess the catalytic activity of many kinases in parallel. HT-KAM relies on collections of biologic peptide probes that are computationally derived from PhosphoAtlas (Olow and Chen et al., Cancer Research 2016) and are physically used as combinatorial sensors to measure the activity of kinase enzymes in biologic extracts. The HT-KAM system provides access to a vast, untapped resource of meaningful measurements, whether readouts are interpreted irrespective of which enzymes phosphorylate which probes, or analyzed to convert global phospho-signatures into functional profiles of kinase activities. Kinome maps reveal how signaling networks are rewired by drug interventions in the context of different cellular backgrounds or exogenously mutated proteins/pathways, and provide insight into potentially targetable pathways. We previously successfully showed that the HT-KAM platform identifies new, actionable kinases responsible for intrinsic or acquired targeted-therapy resistance in BRAFV600E colorectal cancer cells and melanoma tumors from patients. To study TNBC, we used a 615-peptide sensor library that supports the mapping of >100 kinases and represents >900 functional kinase-substrate interactions relevant to tumor biology, and matches hundreds of druggable components of signaling circuits. We characterized the phospho-catalytic signatures of 10 TNBC cell lines, either untreated or treated with chemotherapeutic drugs (5-FU, carboplatin, doxorubicin) at IC50 concentrations. We also included a panel of 8 luminal BC cells. Based on our advanced preliminary data, we anticipate showing a comprehensive map of the oncogenic kinome of TNBC, and how it rewires its signaling network in response to chemotherapies. The most hyperactive and conserved pathways establish a priority map of strategic kinase hot spots to explore as therapeutic candidates. We find that chemotherapy-induced kinases such as SRC, AKT, CHEK, or regulating NFkB or PARP pathways can be inhibited to improve therapeutic sensitivity in TNBC. Citation Format: Jean-Philippe F. Coppe, Pan Bo, Carolien van der Borden, Nina Koemans, Changjun Wang, Denise Wolf, Christina Yau, Sietske Bakker, Marij Hartog, Miki Mori, Ana Ruiz-Saenz, Zhongzhong Chen, Aleksandra Olow, Pei Rong Evelyn Lee, Laura van 't Veer. Systematic identification of the actionable kinase dependencies of chemotherapy-resistant triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 956.

Radiation (RT) is critical to the treatment of high-grade gliomas (HGGs) but cures remain elusive. The BRAF mutation V600E is critical to the pathogenesis of 10-20% of pediatric gliomas, and a small proportion of adult HGGs. Here we aim to determine whether PLX4720, a specific BRAF V600E inhibitor, enhances the activity of RT in human HGGs in vitro and in vivo. Patient-derived HGG lines harboring wild-type BRAF or BRAF V600E were assessed in vitro to determine IC50 values, cell cycle arrest, apoptosis and senescence and elucidate mechanisms of combinatorial activity. A BRAF V600E HGG intracranial xenograft mouse model was used to evaluate in vivo combinatorial efficacy of PLX4720+RT. Tumors were harvested for immunohistochemistry to quantify cell cycle arrest and apoptosis. RT+PLX4720 exhibited greater anti-tumor effects than either monotherapy in BRAF V600E but not in BRAF WT lines. In vitro studies showed increased Annexin V and decreased S phase cells in BRAF V600E gliomas treated with PLX4720+RT, but no significant changes in β-galactosidase levels. In vivo, concurrent and sequential PLX4720+RT each significantly prolonged survival compared to monotherapies, in the BRAF V600E HGG model. Immunohistochemistry of in vivo tumors demonstrated that PLX4720+RT decreased Ki-67 and phospho-MAPK, and increased γH2AX and p21 compared to control mice. BRAF V600E inhibition enhances radiation-induced cytotoxicity in BRAF V600E-mutated HGGs, in vitro and in vivo, effects likely mediated by apoptosis and cell cycle, but not senescence. These studies provide the pre-clinical rationale for clinical trials of concurrent radiotherapy and BRAF V600E inhibitors.

Pediatric low-grade gliomas (PLGG) constitute the most common group of central nervous system tumors in children. Viewed as a chronic disease, ideal therapy for PLGG should carry limited side effects that can be best achieved through selective, personalized therapy. Despite the known heterogeneity of PLGGs and characterized driver mutations most children are still treated with standard chemotherapy protocols as first line therapy. Alteration of the BRAF/MEK/MAPK pathway is the hallmark of PLGG and mTOR activation has been documented in the majority of these tumors. This study investigates combinations of MEK1/2, BRAFV600E and mTOR inhibition in PLGGs carrying specific genetic alterations of the MAPK pathway. We used human glioma cell lines containing BRAFV600E (AM38, DBTRG, BT40), or wild-type BRAF (SF188, SF9427, SF9402) and isogenic systems of KIAA1549:BRAF-expressing NIH3T3 cells and BRAFV600E-expressing murine brain tumors. Signaling inhibitors included everolimus (mTOR), PLX4720 (BRAFV600E), and AZD6244 (MEK1/2). Cell cycle distribution and apoptosis were assessed using flow cytometry; proliferation was determined using an ATP-based assay (CellTiter-Glo). In vivo activity of these inhibitors was assessed in the BT40 PLGG xenograft mouse model. In BRAFV600E tumors, the combination of MEK inhibitor with either mTOR or BRAFV600E specific inhibitor is superior to monotherapy and to combination of BRAF and mTOR inhibitors in reducing cell viability reduction and arresting cell cycle progression. In BRAFWT pediatric gliomas, everolimus + AZD6244 is superior to either agent alone. KIAA1549:BRAF-expressing tumors display marked sensitivity to MEK1/2 inhibition alone. In vivo studies in the PLGG xenograft model BT40 showed the greatest survival advantage in mice treated with combination of AZD6244 + PLX4720 or AZD6244 + everolimus compared with respective monotherapies (p < 0.01).

Tumor-infiltrating inflammatory cells comprise a major part of the stromal microenvironment and support cancer progression by multiple mechanisms. High numbers of tumor myeloid cells correlate with poor prognosis in breast cancer and are coupled with the angiogenic switch and malignant progression. However, the specific roles and regulation of heterogeneous tumor myeloid populations are incompletely understood. CSF-1 is a major myeloid cell mitogen, and signaling through its receptor CSF-1R is also linked to poor outcomes. To characterize myeloid cell function in tumors, we combined confocal intravital microscopy with depletion of CSF-1R-dependent cells using a neutralizing CSF-1R antibody in the mouse mammary tumor virus long-terminal region-driven polyoma middle T antigen breast cancer model. The depleted cells shared markers of tumor-associated macrophages and dendritic cells (M-DCs), matching the phenotype of tumor dendritic cells that take up antigens and interact with T cells. We defined functional subgroups within the M-DC population by imaging endocytic and matrix metalloproteinase activity. Anti-CSF-1R treatment altered stromal dynamics and impaired both survival of M-DCs and accumulation of new M-DCs, but did not deplete Gr-1(+) neutrophils or block doxorubicin-induced myeloid cell recruitment, and had a minimal effect on lung myeloid cells. Nevertheless, prolonged treatment led to delayed tumor growth, reduced vascularity, and decreased lung metastasis. Because the myeloid infiltrate in metastatic lungs differed significantly from that in mammary tumors, the reduction in metastasis may result from the impact on primary tumors. The combination of functional analysis by intravital imaging with cellular characterization has refined our understanding of the effects of experimental targeted therapies on the tumor microenvironment.

High-grade gliomas (HGGs) are aggressive brain tumors in children, with 5-year survival rates

To assess poly (ADP-ribose) polymerase (PARP) inhibitor MK-4827 together with radiation for the treatment of neuroblastoma.Clonogenic survival assays were used to assess MK-4827, radiation and combination thereof in four neuroblastoma cell lines. In vivo efficacy was tested in a murine xenograft model of metastatic neuroblastoma. In vivo targeted inhibition and biological effects included measurement of cleaved caspase-3, γ-H2AX, and Ki 67 by immunohistochemistry (IHC) and poly-ADP-ribose by Enzyme-Linked Immunosorbent Assay.Treatment of neuroblastoma cell lines reduced clonogenicity and resulted in additive effects with radiation. In vivo treatment with MK-4827 and radiation prolonged survival (p0.01) compared to single modalities. In vivo superiority of MK-4827 plus radiation was further documented by significant elevations of cleaved caspase-3 and γ-H2AX in tumors from the combination group compared to single modality cohorts.Combination of MK-4827 and radiation might provide effective therapy for children with high-risk neuroblastoma.

Background/aimTo assess poly (ADP-ribose) polymerase (PARP) inhibitor MK-4827 together with radiation for the treatment of neuroblastoma.Materials and methodsClonogenic survival assays were used to assess MK-4827, radiation and combination thereof in four neuroblastoma cell lines. In vivo efficacy was tested in a murine xenograft model of metastatic neuroblastoma. In vivo targeted inhibition and biological effects included measurement of cleaved caspase-3, γ-H2AX, and Ki 67 by immunohistochemistry (IHC) and poly-ADP-ribose by Enzyme-Linked Immunosorbent Assay.ResultsTreatment of neuroblastoma cell lines reduced clonogenicity and resulted in additive effects with radiation. In vivo treatment with MK-4827 and radiation prolonged survival (p

Introduction: Pediatric low-grade gliomas (PLGG) constitute the most common group of central nervous system tumors in children. Although they exhibit rare malignant transformation and relatively slow growth, PLGGs pose great morbidity. Viewed as a chronic disease, ideal therapy for PLGG should carry limited side effects that can be best achieved through selective, personalized therapy. Despite the known heterogeneity of PLGGs and characterized driver mutations that together offer the perfect, timely platform for personalized approaches to therapy, most children are still treated with standard chemotherapy protocols in a “one treatment fits all” approach. Recent genomic studies have defined recurrent, diverse alterations driving WHO grade 1 and 2 PLGGs. Activation of mitogen protein kinase (MAPK) and phosphatidylinositol-3′ kinase (PI3K) in PLGGs suggests the potential efficacy of agents that target these key growth regulatory pathways. BRAF, a kinase within the MAPK pathway, is activated by missense mutation (V600E) in ∼20% of grade 2-4 pediatric gliomas or KIAA1549-BRAF fusion in ∼60% of pilocytic astrocytomas. In addition to RAS/MEK/MAPK/ERK, the PI3K/AKT/mTOR signaling cascade is activated in ∼half of PLGGs. We hypothesize that knowledge of individual tumor BRAF genotype can guide selection of PI3K/MAPK inhibitors as single agents and in combinations, to maximize efficacy of therapy and overcome innate and acquired resistance to targeted agents. Methods: We used human glioma cell lines containing BRAFV600E (AM38, DBT-RG, BT40), or wild-type BRAF (BRAFWT; SF188, SF9427, SF9402) and isogenic systems of KIAA1549:BRAF-expressing NIH3T3 cells and BRAFV600E-expressing murine brain tumors. Signaling inhibitors included everolimus (mTOR), PLX4720 (BRAFV600E), and AZD6244 (MEK). Cell cycle distribution and apoptosis were assessed using flow cytometry; proliferation was determined using an ATP-based assay (CellTiter-Glo). In vivo activity of these inhibitors was assessed in the BT40 PLGG xenograft mouse model. Results: In BRAFV600E cells, combination of PLX4720+everolimus and PLX4720+AZD6244 exhibited significantly greater effects on cell viability, apoptosis, and cell cycle than respective single agents. In BRAFWT cells, everolimus+AZD6244 was superior compared to respective monotherapies. Similar results were found using isogenic murine cells. In KIAA1549:BRAF fusion cells, MEK inhibition reduced cell viability and S-phase content, effects that were modestly augmented by mTOR inhibition. In vivo experiments in the PLGG xenograft model BT40 showed the greatest survival advantage in mice treated with combination of AZD6244+PLX4720 or AZD6244+everolimus compared with respective monotherapies (p Conclusions: For BRAFV600E tumors, combination of everolimus+MEK or PLX4720+MEK inhibitors is equally efficacious and superior to single agents, and the choice between these two might be dictated by clinical tolerability. For BRAFWT PLGGs, combination of everolimus+MEK inhibitor is superior to single agent therapies. Optimal treatment of KIAA1549:BRAF-expressing PLGGs is still under investigation as single agent MEK inhibition is extremely efficacious but mTOR inhibition may contribute to greater overall anti-neoplastic effects. Citation Format: Aleksandra K. Olow, Sabine Mueller, Xiaodong Yang, Rintaro Hashizume, William Weiss, Adam C. Resnick, Angela J. Sievert, Mitchel S. Berger, Nalin Gupta, David C. James, Daphne A. Haas-Kogan. Targeting PI3K in personalized treatment of BRAF-mutated pediatric low-grade gliomas. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B42.

10060 Background: Neuroblastoma (NB) is the most common solid extra-cranial tumor of childhood. 131I-mIBG is a targeted radiopharmaceutical for patients with advanced NB and is one of the most active agents in this treatment resistant population. To date, no clinically relevant biomarkers of response or toxicity have been reported in patients treated with 131I-mIBG. Methods: Patients with relapsed or refractory NB who received 131I-mIBG at UCSF were eligible to participate in this prospective correlative study. Blood samples were obtained at baseline and 72 hours after 131I-mIBG infusion. We quantified a panel of biomarkers shown to be effected in patients receiving other forms of radiation, including: serum amylase; plasma Flt-3 ligand; plasma monocyte colony stimulating factor (MCSF); and CDKN1A mRNA in mononuclear cells. Extent of modulation of each marker was evaluated using paired t-tests at hour 0 and 72. We assessed potential differential modulation between groups based on response (PR/CR vs SD/PD), toxicity (grade 3 non-hematologic toxicity; grade 4 neutropenia or thrombocytopenia) or administration of combination therapy (131I-mIBG alone vs in combination with vincristine/irinotecan or vorinostat) using unpaired t-tests. Results: 26 patients (21 male; median age 7 years) participated and received a median 131I-mIBG dose of 18 mCi/kg. 16 patients received 131I-mIBG in combination. We observed robust modulation of amylase (median fold increase from baseline 3.1; p 131I-mIBG had more robust modulation of Flt-3 ligand (mean difference 296 pg/mL; p=0.02) compared to single agent 131I-mIBG. No correlations with clinical response or toxicity were found. Conclusions: Amylase, Flt-3 ligand, and CDKN1A mRNA all show robust modulation after 131I-mIBG treatment. Patients receiving 131I-mIBG in combination demonstrated the largest changes in plasma Flt-3 ligand. In this initial analysis, no associations were found between response or toxicity and the proposed biomarkers.