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Introduction: Triple Negative Breast Cancer (TNBC) is an aggressive disease with a poor prognosis that accounts for 10-20% of breast cancer cases worldwide. Intra-tumoral heterogeneity and tumor cell plasticity are thought to contribute to drug resistance in TNBCs. Our work aims to: 1) precisely identify the intra-tumoral heterogeneity in cellular states present within TNBC, and 2) test whether drugs can block or initiate plasticity between subpopulations to improve drug sensitivity. We hypothesize that treatment(s) induce cellular plasticity, thus causing cells to shift into resistant states that persist until treatment is removed. We further hypothesize that these resistant subpopulations give rise to new tumor outgrowths once the treatment stops. Methods: To test this, we are treating TP53-/- Genetically Engineered Mouse Model (GEMM) syngeneic transplant tumors of the basal-like TNBC phenotype with the chemotherapeutic doublet of carboplatin/paclitaxel, and targeted agents implicated in plasticity including the MEK inhibitor trametinib, a chromatin remodeling inhibitor I-BET151, and the dihydroorotate dehydrogenase inhibitor brequinar. To identify cellular subpopulations and examine their response to treatment, we performed both in vivo and in vitro drug sensitivity testing, as well as gene expression profiling using single cell RNA-sequencing (scRNAseq). Results: We have identified clear intra-tumoral heterogeneity with at least 6 distinct cell states present, including basal, mesenchymal/claudin-low, and proliferative subpopulations in vivo in most TNBC GEMM models. We performed 18 individual scRNAseq experiments on the TP53-/- 2225L GEMM transplant line with the aforementioned treatments and untreated controls in triplicate and compared subpopulation frequencies in treated versus untreated tumors. Notably, treatment with trametinib and brequinar caused the rise of two rare subpopulations (i.e. 1% to 3-4% of total tumor cells) that express genes consistent with previously described drug-tolerant persisters (DTP), which we have called “Epithelial-DTP” (Tacstd2, Krt6a, and Cryab enriched) and “Mesenchymal-DTP” (Snai2 and Sca-1 enriched). A gene signature generated from the Epithelial-DTP subpopulation predicted poor patient outcomes in neoadjuvant chemotherapy treated TNBC patients. Further, in TNBC patient-derived xenografts (PDX), these two DTP subpopulations are also present and induced by treatment to an even greater frequency. Ongoing experiments include the use of fluorescence-activated cell sorting to isolate and functionally test the tumor-initiating capabilities of these two rare cell subpopulations. In addition, many experiments are underway to identify means to therapeutically target these DTP cells, with these results to be presented. Ultimately, identifying these rare drug resistant subpopulations, and identifying means to eradicate them, could vastly improve therapeutic regimens and outcomes for patients with TNBCs. Citation Format: Cherise R. Glodowski, Kevin R. Mott, Denis Okumu, Michael P. East, Timothy C. Elston, Gary L. Johnson, Charles M. Perou. PD4-09 Single cell RNA-sequencing identifies intra-tumoral cellular heterogeneity and drug-induced subpopulation shifts in Triple Negative Breast Cancer mouse models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-09.

Introduction: Translational research is essential to the success of every cancer center. Increasingly doctors ask patients to donate tissue to study all aspects of the disease to improve cancer treatments and quality of life. Patients are key stakeholders and partners in the success of the translational research process. The University of North Carolina (UNC), Lineberger Comprehensive Cancer Center (LCCC) researchers would like to increase the number of patients that donate tissue to support research and value patients who donate. Securing the right tissues at the right time, and processing and storing them properly is essential for research that leads to breakthrough discoveries. Purpose: LCCC supports the inclusion of patient advocates in all cancer center research. Therefore, the UNC Breast SPORE patient advocates initiated a program called Patients and Researchers Together (PART) to ensure patients work closely with researchers to drive the best research forward. The PART-Tissue program aims to develop a process to increase the donation and usage of patient donated tissues. This program is built on significant patient engagement that leads to a bi-directional patient and researcher PARTnership. We believe patients will be more willing to donate tissue if they are informed about the research process, understand the impact research has to improve patient’s lives, and are valued as PARTners of LCCC. Objectives: The first objective is to promote the excellence of LCCC translational research to all stakeholders, including patients and the community. Second; create a patient-centered tissue donation program that will meet researcher needs across LCCC, and Third; ensure the tissue collection process is primarily focused on recognizing patient’s voluntary contributions while minimizing patient burden. Methods: We first created a multi-stakeholder committee to ensure the participation of key stakeholders in all activities. We developed a process to identify relevant materials to assist in tissue donation at LCCC. The project focused on a Lineberger breast cancer tissue collection protocol (LCCC9819). We discussed patient, researcher and clinician/surgeon needs that reflect the vision of the PART program. We identified the need for several types of informational materials for patients, developed them with multi-stakeholder involvement and evaluated their accuracy, readability, understandability, and health literacy. We assessed the current and future needs of LCCC researchers by conducting interviews and surveys. We also engaged patient advocates as PARTners throughout this project. Results: We initiated the PART-Tissue program in August 2020 and we have engaged multiple stakeholders throughout the project. We evaluated LCCC9819 from the patient, research and clinical perspectives. We identified a series of informational materials needed for patients to introduce them to the value of tissue donation and to introduce them to the LCCC9819 protocol. We developed two introductory materials and a study summary. We conducted interviews and developed a survey to collect information about researcher needs at LCCC. In pilot interviews, we noticed a high need for fresh tissue, which requires real-time coordination between clinicians, clinical research coordinators, surgeons, pathologists and researchers. We also developed a website to connect directly with patients and the community about the importance of tissue donation that can lead to discoveries in cancer risk reduction and treatment. The goal is to develop a process where patients feel encouraged to donate tissue samples and are appreciated as partners with researchers who use the tissue to improve the lives of cancer patients. Citation Format: Patricia A. Spears, Vernal Branch, Jennifer A. Potter, Missy Van Lokeren, Nasrin H. Babadi, Adriana S. Beltran, Laurie Betts, Coleen Crespo, Barbara Dean, Amy L. Garrett, Laura T. Jensen, Gary L. Johnson, Hayley Morris, Philip Spanheimer, Charles M. Perou, Lisa A. Carey, H. Shelton Earp. Patients and Researchers Together (PART); a patient-centered tumor tissue collection PARTnership between patients and researchers to increase tissue donations for breast cancer research [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-15-01.

We have previously identified two tumor specific molecular subtypes in pancreatic ductal adenocarcinoma (PDAC), basal-like and classical. Although basal-like tumors are found in less than 20% of patients, patients with basal-like tumors have a worse prognosis and are largely resistant to FOLFIRINOX chemotherapy. To identify targetable basal-like subtype specific vulnerabilities in PDAC, we utilized Multiplexed kinase Inhibitor Beads and Mass Spectrometry (MIB-MS) to profile the kinome in patient derived xenograft (PDX) models that recapitulate the tumor subtypes found in patients. MIB-MS results show that kinase expression is significantly associated with molecular subtype. Notably, basal-like tumors show increased expression of receptor tyrosine kinases (RTKs) including EGFR, MET, and IGF1R and MAP kinase members MEK2 and BRAF indicating activation of the MAP kinase signaling cascade. These RTKs including EGFR are also differentially expressed in primary patient tumors suggesting the true efficacy of EGFR and other kinase inhibitors in basal-like tumors may have been masked by the larger proportion of patients (>80%) with unresponsive classical tumors. Furthermore, inhibition of MAP kinase signaling with the MEK inhibitor trametinib results in kinome reprogramming exclusive to classical tumors. MIB-MS profiling reveals upregulation of TAOK3 and MEK3/6 indicating activation of p38 MAP kinase signaling as a classical subtype specific compensatory mechanism against MEK inhibition. Overall, these results present actionable basal-like subtype specific kinase targets by defining a novel subtype specific kinome in PDAC. Precision approaches in clinical trials are needed to determine if new and previously thought to be disappointing kinase inhibitors may be efficacious in patients with basal-like tumors. Citation Format: Yi Xu, Ashley B. Morrison, Silvia G. Herrera, Michael P. East, Gary L. Johnson, Jen Jen Yeh. Proteomic profiling reveals subtype specific kinase expression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR011.

Glioblastoma (GBM) is a malignant brain tumor that has proven difficult to treat, despite expressing promising targets such as EGFRvIII. EGFRvIII, a mutant version of the epidermal growth factor receptor (EGFR), is constitutively active and not present in normal brain cells. The tumor specificity of EGFRvIII and the frequent EGFR amplification seen in GBM make EGFR a potentially attractive therapeutic target; however, clinical studies have shown little to no efficacy for EGFR tyrosine kinase inhibitors (TKI). One reason for this lack of efficacy may be adaptive resistance. We used RNA sequencing and multiplexed inhibitor beads with mass spectrometry (MIB-MS) to study the transcriptomes and kinomes of genetically engineered mouse astrocytes to investigate this resistance and identify potential targets for dual inhibition. Out of 329 kinases detected by MIB-MS, 76 were differentially expressed between cells with Cdkn2a deletion (“C”) and cells that also overexpressed EGFRvIII (“CEv3”). Thirty-four of these kinases were overexpressed in the CEv3 cells relative to the parental C cells (log2 fold change of 5.6, p Citation Format: Erin Smithberger, Abigail K. Shelton, Ryan E. Bash, Madison K. Butler, Alex R. Flores, Allie Stamper, Steven P. Angus, Michael P. East, Gary L. Johnson, Michael E. Berens, Frank B. Furnari, Ryan Miller. Glioblastoma growth is suppressed dual inhibition of EGFR and CDK6 kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1857.

Glioblastoma (GBM) is a devastating primary brain tumor with Citation Format: Abigail K. Shelton, Erin Smithberger, Madison Butler, Allie Stamper, Ryan E. Bash, Steve P. Angus, Michael P. East, Gary L. Johnson, Michael E. Berens, Frank B. Furnari, Ryan Miller. Acquired resistance to targeted inhibitors in EGFR-driven glioblastoma: Identification of dual kinase targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3248.

Targeted therapeutics that are initially effective in cancer patients nearly invariably engender resistance at some stage, an inherent challenge in the use of any molecular-targeted drug in cancer settings. In this study, we evaluated resistance mechanisms arising in metastatic melanoma to MAPK pathway kinase inhibitors as a strategy to identify candidate strategies to limit risks of resistance. To investigate longitudinal responses, we developed an intravital serial imaging approach that can directly visualize drug response in an inducible RAF-driven, autochthonous murine model of melanoma incorporating a fluorescent reporter allele (tdTomatoLSL). Using this system, we visualized formation and progression of tumors in situ, starting from the single-cell level longitudinally over time. Reliable reporting of the status of primary murine tumors treated with the selective MEK1/2 inhibitor (MEKi) trametinib illustrated a time-course of initial drug response and persistence, followed by the development of drug resistance. We found that tumor cells adjacent to bundled collagen had a preferential persistence in response to MEKi. Unbiased transcriptional and kinome reprogramming analyses from selected treatment time points suggested increased c-Kit and PI3K/AKT pathway activation in resistant tumors, along with enhanced expression of epithelial genes and epithelial-mesenchymal transition downregulation signatures with development of MEKi resistance. Similar trends were observed following simultaneous treatment with BRAF and MEK inhibitors aligned to standard-of-care combination therapy, suggesting these reprogramming events were not specific to MEKi alone. Overall, our results illuminate the integration of tumor–stroma dynamics with tissue plasticity in melanoma progression and provide new insights into the basis for drug response, persistence, and resistance. Significance: A longitudinal study tracks the course of MEKi treatment in an autochthonous imageable murine model of melanoma from initial response to therapeutic resistance, offering new insights into the basis for drug response, persistence, and resistance. Cancer Res; 78(2); 542–57. ©2017 AACR.

Pancreatic adenocarcinoma (PDAC) remains an extremely lethal disease with few effective therapeutic options. Targeted therapy development has remained largely unsuccessful due to complex tumor heterogeneity and better molecular indicators of response are urgently needed. We have previously identified two distinct molecular tumor subtypes in PDAC, basal-like and classical, which are prognostic and dictate response to chemotherapy. We have also developed a patient derived xenograft (PDX) mouse model that maintains the integrity of patient tumor biology and recapitulates primary tumor subtypes in vivo. Here, we aim to utilize multiplexed kinase inhibitor beads and quantitative mass spectrometry (MIB-MS) to determine baseline kinase expression and adaptive kinome responses to therapy in PDAC PDX tumors. A total of 381 kinases were identified across all samples and tumor specific kinases were isolated by alignment to human peptides. Distinct kinase expression profiles were observed for basal-like and classical tumors. Basal-like tumors showed high expression of kinases involved in receptor tyrosine kinase activity, MAP kinase activity, and clathrin-dependent endocytosis. Classical subtype tumors showed increased expression of kinases involved in metabolism and cytoskeletal regulatory kinases. Differential protein expression was further validated by differential gene expression using RNAseq. MIB profiling of kinome response to EGFR inhibition by erlotinib indicated kinome reprogramming with upregulation of microtubule associated kinases and MAP kinase family members. Overall, these results suggest that basal-like and classical subtype tumors exhibit distinct kinome profiles that present possible subtype specific vulnerabilities to kinase inhibition. Stratifying kinase inhibitor therapies based on tumor subtype could improve response to targeted therapies in PDAC. Citation Format: Yi Xu, Michael East, Ashley Morrison, Gabriela Herrera, Laura Peng, Gary Johnson, Jen Jen Yeh. Proteomic profiling reveals subtype specific kinase expression in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-079.

High-dose, single-drug treatment is unsuccessful at suppressing metastasis due to cellular heterogeneity and feedback activation. Here we demonstrate that targeting driver network signaling capacity by limited inhibition at multiple nodes is a more effective anti-metastatic strategy. A physiological metastasis suppressor, Raf Kinase Inhibitory Protein, decreases the activity of multiple kinases in the stress-regulated MAP kinase (MAPK) network and reduces the output to metastatic transcription factor BACH1 that upregulates motility-related genes. This effect is mimicked by a low-dose four-drug cocktail (4D-MAPKi) that blocks metastatic colonization in mouse breast cancer models and increases survival. Drug treatment and network modeling show that 1) multi-kinase inhibition is required to suppress signaling output across cells with different MAPK network topologies, and 2) restricting inhibition of individual kinases prevents threshold activation of other compensatory kinase networks. This low-dose multi-drug approach to decrease signaling capacity of a driver network represents a transformative, clinically-relevant strategy for anti-metastatic treatment. Citation Format: Ali Ekrem Yesilkanal, Dongbo Yang, Payal Tiwari, Alan U. Sabino, Jiyoung Lee, Xiao-He Xie, Siqi Sun, Christopher Dann, Ethan Steinberg, Timothy Stuhlmiller, Casey Frankenberger, Elizabeth Goldsmith, Gary L. Johnson, Alexandre F. Ramos, Marsha Rosner. Limited inhibition of multiple nodes in a driver network blocks metastasis [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 5038.

Background: Cabozantinib, an oral FDA approved multi-receptor tyrosine kinase inhibitor, was tested in our preclinical mouse model of PN. After finding significant reduction of tumor number and size in cabozantinib treated versus control mice, we sought to translate these findings to a phase 2 human study. Here we report the activity of cabozantinib in adolescents and adults with NF1-associated PN. Methods: A multicenter, nonrandomized phase 2 trial (NCT02101736) of cabozantinib in subjects ≥16 years with NF1 and either progressive or clinically significant inoperable PN was performed by the NFCTC (NF-105). The primary study aim was volumetric response of the target PN determined by MRI read centrally. Cabozantinib was administered continuously for up to 24 cycles, each cycle was 28 days. The starting dose was 40 mg once daily with planned escalation to a target dose of 60 mg once daily after 2 cycles. Dose reductions for toxicity were allowed to 20 mg once daily. Partial response (PR) was defined as ≥20% reduction in tumor volume from baseline. Subjects were considered evaluable for response if they completed ≥1 cycle of therapy and had a follow-up MRI. Success was defined as ≥25% of subjects achieving and maintaining a PR after 12 cycles without significant toxicity. Investigation of the impact of cabozantinib on the PN kinome network was performed on murine samples. Results: Twenty-three subjects enrolled; 21 subjects (median age 22 years) were evaluable for toxicity (2 noted to be ineligible before receiving study drug) and 19 subjects (median age 23 years) were evaluable for response (1 subject withdrew during cycle 1 and 1 was found ineligible after starting study drug). Baseline median tumor size was 557 mL (range 57-2954 mL). Among the evaluable patients, 8 (42%) met criteria for PR by cycle 12. Median change in tumor volume was -15.2% (range +2.2% to -36.9%). No subject had PN progression on treatment; maximal tumor response was not achieved until at least 18 cycles in 6/8 responders. A significant portion of patients underwent dose reductions or discontinued cabozantinib due to low grade adverse events (AE) that impaired quality of life; however, 3 responders reduced to 20 mg maintained or improved their response at this dose. The most common AEs (any grade) in ≥10 patients included gastrointestinal toxicity, hypothyroidism, fatigue and palmar plantar erythrodysesthesia (PPE). Eleven grade 3 AEs occurred in 8 patients, mainly PPE (n=4) and hypertension (n=2); no grade 4 or 5 AEs occurred. Analysis of kinase activity in murine PN treated with cabozantinib showed significantly decreased activity of AXL, MERTK and MET, known cabozantinib targets, but also of DDR1 and DDR2. Conclusions: Cabozantinib demonstrates considerable clinical activity for PN with a radiographic response rate of 42%. Although there were few severe AEs, low grade toxicities impacted the willingness of many subjects to continue treatment. Quantitative kinome analysis revealed that inhibition of DDR1, DDR2, AXL, MERTK and MET might underpin the therapeutic responses seen in these patients. Lower doses of cabozantinib may be optimal for the NF1 population and still lead to therapeutic response. This trial is now enrolling a pediatric cohort of children aged 3 to 15 years. Supported by DOD Award W81XWH-12-1-0155 and Exelixis Citation Format: Chie-Schin Shih, Jaishri Blakeley, D. Wade Clapp, Amy E. Armstrong, Pam Wolters, Eva Dombi, Gary Cutter, Nicole J. Ullrich, Jeffrey Allen, Roger Packer, Stewart Goldman, David H. Gutmann, Scott Plotkin, Tena Rosser, Kent Robertson, Brigitte Widemann, Steven Rhodes, Steven Angus, Gary Johnson, Bruce Korf, Michael J. Fisher. Treatment of neurofibromatosis type 1 (NF1)-related plexiform neurofibromas (PN) with cabozantinib (XL184): A Neurofibromatosis Clinical Trials Consortium Phase II trial [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 CT233.

Glioblastoma (GBM) is a devastating primary brain tumor with limited treatment options. Extensive molecular characterization has revealed two particularly frequent mutations: CDKN2A deletion (50-60%) and EGFR (40-50%). EGFRvIII (~35%) is a constitutively active truncation mutant with exons 2-7 deleted. EGFR is a particularly attractive therapeutic target due to frequent activating mutations, such as EGFRvIII, and ready availability of multiple targeted inhibitors. Several EGFR tyrosine kinase inhibitors (TKI) have failed clinically, due in part to acquired resistance. To mechanistically examine this type of resistance, we used genetically-engineered mouse astrocytes harboring homozygous deletions of Cdkn2a, as well as EGFRvIII (CEv3). CEv3 astrocytes were made intrinsically resistant to the EGFR TKI gefitinib or erlotinib via long-term exposure, both in vitro and in vivo. We found that long-term gefitinib or erlotinib exposure conferred variable levels of cross resistance to a panel of second- and third-generation EGFR TKI (ΔIC50 1.12-36.1-fold), relative to non-resistant parent lines. We have previously shown that dynamic kinome reprogramming may be responsible for TKI resistance in glioblastoma. Therefore, we used a chemical proteomics method, multiplexed inhibitor beads and mass spectrometry (MIB-MS), to examine changes in the expressed and functional kinome, in both the presence or absence of one of several EGFR TKI known to penetrate the blood-brain barrier. Additionally, we performed RNA sequencing (RNA-seq) to inspect transcriptomic alterations in response to these drugs. RNA-seq showed that resistant CEv3 mouse astrocytes clustered separately from their non-resistant in vitro and in vivo counterparts. Acquired resistance also induced transcriptome alterations governing cellular metabolism, including upregulation of metabolic pathways and downregulation of RNA processing genes. Importantly, the kinase transcriptome was rewired, as 67 kinases were differentially expressed across parental and resistant cell lines (Q Citation Format: Abby Shelton, Erin Smithberger, Madison Butler, Alex Flores, Ryan Bash, Steve Angus, Noah Sciaky, Harshil Dhruv, Gary L. Johnson, Michael E. Berens, Frank Furnari, C. Ryan Miller. Dynamic kinome targeting reveals kinases involved in acquired resistance to tyrosine kinase inhibitors in EGFR-driven glioblastomas [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 331.

Glioblastoma (GBM) is an aggressive brain tumor with few effective treatments. Epidermal growth factor receptor (EGFR) is frequently amplified and mutated in GBM, leading to trials of several EGFR tyrosine kinase inhibitors, but none have proven successful. One potential reason for failure is acquired resistance, particularly acute, adaptive responses in the kinome. To study this adaptive resistance mechanism, we used RNA-seq and multiplex inhibitor bead/mass spectrometry (MIB-MS) to analyze transcriptomes and kinomes of genetically-engineered murine astrocytes with genotypes commonly seen in human GBM. We previously showed that 38% (86 of 228) of the expressed kinome varied among a panel of genetically diverse murine astrocytes harboring Cdkn2a deletion (C) plus Pten deletion (CP), wild-type human EGFR (CE) or EGFRvIII (CEv3) overexpression, or both overexpressed EGFRvIII and Pten deletion (CEv3P). Pairwise genotype comparisons revealed multiple differentially activated kinases, including Pdgfrb, Fgfr2, Lyn, Ddr1, and several Ephrin family members. We further investigated these potential targets for dual therapy with EGFR TKI by examining the transcriptional response of cultured astrocytes at 4, 24, and 48 hours after 3 μM afatinib. Afatinib induced no kinome changes in C and only 3 kinases (Fn3k, Prkg2, and Syk) were altered in CP astrocytes. Despite similar baseline gene expression profiles, CE astrocytes overexpressing wild-type EGFR responded significantly differently than C astrocytes without. Five kinases (Dclk1, Epha3, Epha7, Fgfr3, and Prkg1) were induced, while 14 were repressed. Six were similarly repressed in CEv3 (Bub1, Nek2, Pask, Plk4, Prkcb, and Vrk1). Whereas the kinase transcriptome response was blunted in C, CP, and CE astrocytes, afatinib induced altered expression of significantly more kinases in CEv3 (82) and CEv3P cells (49). One particularly attractive target in CEv3 astrocytes was Epha4, which afatinib induced >40-fold. Dual inhibition of EGFRvIII and Epha4 kinases may thus provide an opportunity for more effective targeted therapy. Citation Format: Erin Smithberger, Abigail K. Shelton, Madison K. Butler, Alex R. Flores, Ryan E. Bash, Steven P. Angus, Noah Sciaky, Harshil D. Dhruv, Gary L. Johnson, Michael E. Berens, Frank B. Furnari, C. Ryan Miller. Dynamic kinome profiling of EGFRvIII-driven murine astrocyte models of glioblastoma reveals targets for dual kinase inhibitor therapy [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 3019.

Metastatic progression of tumors is the major cause of death in patients with triple-negative breast cancer (TNBC). However, since metastasis is a multistep process, unraveling its complexity is a major challenge. One effective way of tackling this question is to study natural blockers of the metastatic process, metastasis suppressors, and identify the mechanisms by which they regulate metastasis. Raf kinase inhibitory protein (RKIP), a protein that regulates kinase activity, is a suppressor of TNBC metastasis. Although RKIP inhibits the activity of key kinases such as Raf-1, GRK2, NIK/IKK in cultured cells, the kinase targets of RKIP in tumors are not known. To address this question, we used a mass spectrometry approach involving inhibitor-conjugated beads to identify kinases that are downregulated by RKIP in human TNBC xenograft tumors. Our results identified a network of stress kinases targeted by RKIP, including kinases that have not been previously reported as RKIP targets. In order to unravel the effect of this stress network on metastatic gene expression, we investigated genes that correlate with RKIP expression in TCGA breast cancer patient data set. We identified prometastatic genes such as APC and DOCK4 as novel RKIP targets in in vitro and in vivo models of TNBC. We also demonstrated these genes are downstream of the RKIP-stress network. Finally, by using a high-throughput invasion assay, we developed a low-dose multidrug cocktail of small-molecule kinase inhibitors that mimic RKIP's antimetastatic role in TNBCs. Elucidating RKIP function at a systems level reveals the interplay between key metastatic signaling cascades, particularly in relation to cell motility and invasion. Our findings suggest that the low-dose multidrug combination that targets a network of stress kinases is a viable antimetastatic therapy for TNBC patients. Citation Format: Ali Ekrem Yesilkanal, Daniel C. Rabe, Payal Tiwari, Casey Frankenberger, Gary L. Johnson, Marsha Rosner. A novel approach for antimetastatic therapies against TNBC utilizing a physiologic suppressor [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 4179.

The discovery of mutations in the ALK oncogene as the genetic etiology of familial neuroblastoma (NB) and as somatically acquired in 14% of patients with the most aggressive form of the disease has positioned ALK as the major tractable oncogene product for targeted therapy in NB. ALK tyrosine kinase domain mutations are found mostly at three hotspots (F1174, F1245 and R1275), with the F1174* and F1245* mutations conferring broad resistance to crizotinib. We have recently demonstrated that lorlatinib, a novel ATP-competitive macrocyclic ALK inhibitor, overcomes de novo resistance and exerts unprecedented activity as a single agent in patient-derived xenografts (PDXs) harboring F1174L or F1245C mutations, while also inducing durable responses in R1275Q xenografts. The objective of our work was to identify differences in reprogramming of the kinome upon ALK inhibition with crizotinib and lorlatinib. We hypothesize that identification of the targets through which lorlatinib mediates its superior anti-tumor effects will provide insights into the mechanisms by which full-length ALK inhibition abrogates tumorigenesis, activates compensatory pathways, and uncover novel targets to overcome resistance to ALK inhibition. We used Multiplexed Inhibitor Beads coupled with Mass Spectrometry (MIB/MS) to quantitatively measure kinase activity dynamics on a proteomic scale. Three PDX models harboring F1174L (COG-N-453x) or the other two most-common mutations, R1275Q (NB1643) and R1245C (Felix) were treated with either crizotinib (100 mg/kg/day) or lorlatinib (10 mg/kg/day) for 2.5 days or 6.5 days prior to MIB/MS analysis. Lorlatinib more potently inhibited ALK and preferentially inhibited a series of other tyrosine kinases, correlating with the superior efficacy of lorlatinib in pre-clinical studies. Moreover, a number of kinases involved in G2/M cell cycle transition including PLK1, CHEK1 and aurora kinases were significantly inhibited by lorlatinib, proposing that this multi-kinase inhibition is responsible for the robust anti-tumor activity observed in vivo with lorlatinib. We interrogated the role of SHP2, a ubiquitously expressed SH2 domain-containing tyrosine phosphatase recently implicated in the proliferation of receptor-tyrosine kinase driven cancers and shown to have a direct role in the G2/M checkpoint. Inhibition of ALK by treatment with lorlatinib as well as shRNA knockdown of ALK in SY5Y (ALK F1174L, PTPN11 T507K) and NB1643 NB cell lines showed decreased levels of pSHP2. Depletion of SHP2 using CRISPR constructs significantly inhibited cell proliferation in SY5Y, suggesting that SHP2 plays a crucial role in ALK driven NB. Additionally, knockdown of ALK in SY5Y downregulated PLK1 expression and arrested cells in G2/M phase. Whether SHP2 is regulated primarily downstream of ALK or jointly through alternative or compensatory signaling networks warrants further investigation. Citation Format: Smita Matkar, Renata Sano, Colleen Larmour, Kateryna Krytska, Gabriela M. Witek, Mark Gerelus, Tim J. Stuhlmiller, Gary L. Johnson, Yael P. Mosse. Chemical proteomics identifies druggable proteins in ALK-driven neuroblastomas [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 1633.

Basal-like or triple-negative breast cancer, if considered its own disease, would rank as the 5th leading cause of cancer deaths for women in the USA each year with ~10,000 deaths annually. Given the lack of drug-able targets expressed by TNBC tumors, few therapeutic options exist beyond currently utilized cytotoxic therapies, and the overall prognosis for these patients remains poor. The oncogenic PI3K/Akt signaling axis contributes to the dysregulation of many dominant features in breast cancer including cell proliferation, survival, metabolism, motility, and genomic instability. While multiple studies have demonstrated that basal-like or triple-negative breast tumors have uniformly high PI3K/Akt activity, genomic alterations that mediate dysregulation of this pathway in this subset of highly aggressive breast tumors remain to be determined. In this study, we present an integrated genomic analysis based on the use of a PI3K gene expression signature as a framework to analyze orthogonal genomic data in human breast tumors from TCGA (The Cancer Genome Atlas) including RNA expression, DNA copy number alterations, and protein expression. These analyses identified copy number alterations associated with PI3K activity in human breast tumors (n=1,031), including known and potentially novel drivers of PI3K signaling. Data from a genome-wide RNA interference (RNAi) screen were used to discriminate between essential and non-essential genes within identified amplicons. Integrating DNA copy number and RNAi analyses identified amplified SOX4 as a novel and essential regulator of PI3K/Akt signaling. Importantly, by analyzing protein expression data (n=733), we determined that SOX4 amplified tumors have increased activation of pAkt substrates. Moreover, depleting SOX4 levels in HCC38 and HCC1143, basal-like breast cancer cell lines with high PI3K activity and high SOX4 expression, demonstrated significant abrogation of p-Akt levels. This validated our in silico findings and confirmed the role of SOX4 in regulating PI3K pathway activity. To identify the potential mechanism(s) by which SOX4 mediates PI3K pathway activity, we performed genome-wide analyses of these cell lines following SOX4 inhibition. Transcriptomic profiling by RNA-sequencing, protein profiling by Reverse Phase Protein Array (RPPA) and Kinome profiling through multiplexed kinase inhibitor beads and mass spectrometry (MIB-MS) based approach identified TGFBR2, as a potential regulator of SOX4-mediated PI3K/Akt signaling. Further studies will elucidate the mechanism by which SOX4 modulates TGFBR2 expression and drives the oncogenic PI3K/Akt signaling pathway in basal-like breast cancer. Citation Format: Gaurav Mehta, Michael Gatza, Steve Angus, Gary Johnson. Amplification of SOX4 promotes PI3K/Akt signaling in human 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 404.

The most attractive target for personalized medicine of glioblastoma (GBM) remains epidermal growth factor receptor (EGFR), due to its frequency in and specificity for the disease and the number of drugs that target its tyrosine kinase domain. Yet several EGFR tyrosine kinase inhibitors (TKI) have failed clinically, in part due to multiple molecular mechanisms of resistance. The most common oncogenic mutation in GBM is EGFRvIII, while the most common tumor suppressors lost are CDKN2A and PTEN. Mutations in these 3 genes frequently co-occur. To identify potentially attractive kinase targets for use in combination regiments with an EGFR TKI, we used multiplex inhibitor bead/mass spectrometry (MIB-MS) and RNA-seq to examine the baseline kinomes and transcriptomes of non-germline genetically engineered mouse (nGEM) models of GBM, specifically cultured Cdkn2a-null murine astrocytes (C) engineered to harbor human EGFRvIII (CEv3), Pten deletion (CP), or both (CEv3P). Among these 4 lines, 5.2-9.7% of the transcriptome was differentially expressed using DESeq2 at QCEv3> CP>C), suggesting that EGFRvIII and Pten deletions cooperate to induce astrocyte de-differentiation into glioma stem cells (GSC). Principal components analysis showed a significant influence of both EGFRvIII (component 1, 44-48% variance) and Pten (component 2, ~33% variance) status on the transcriptome and kinome. Of the 228 expressed kinases detected using MIB-MS, 86 (38%) were differentially expressed in 1 or more genotypes. Integrated transcriptome and kinome analysis showed that Egfr was significantly over-expressed and hyperactive in EGFRvIII-mutated cells. Akt1 showed a non-expression driven increase in kinase activity in Pten-deleted cells, consistent with known effects of Pten on PI3K signaling. Pairwise genotype comparisons revealed 5-20 additional kinases that were differentially activated. Some, including Pdgfrb, Fgfr2, Lyn, Ddr1, and several members of the Ephrin family, represent potential targets for dual therapy with EGFR TKI. Clinically-curated human GBM patient-derived xenograft (PDX) models matched to CEv3P, CEv3, CP, and C nGEM models will afford comparisons in a patient-based preclinical setting for translational support. Functional kinome analysis using targeted EGFR TKI and MIB-MS in nGEM and PDX will help define the kinase networks required for EGFRvIII-driven GBM pathogenesis and may aid in the identification of novel treatment combinations. Citation Format: Erin Smithberger, Alex R. Flores, Madison K. Butler, Harshil D. Dhruv, Gary L. Johnson, Michael E. Berens, Frank B. Furnari, C. Ryan Miller. Kinome profiling of non-germline, genetically engineered mouse models of glioblastoma driven by Cdkn2a, Egfr, and/or Pten mutations reveals genotype-dependent kinase targets [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 2372.

Multiplexed Inhibitor Beads coupled with quantitative Mass Spectrometry (MIB/MS) allows study of over 60-70% of the expressed kinome simultaneously using lysates from cell lines, mouse tumors and human specimens. Cells or tumors are assessed before and after addition of agonists or inhibitors. Results show a systems kinome reprogramming in response to highly selective inhibitors that creates inhibitor resistance. The activation signatures and changes in the kinome activation profile (kinome reprogramming) are unique for each breast cancer subtype (Luminal, HER2+, Basal-like, Claudin-low) in response to highly selective, clinically relevant kinase inhibitors (MEKinh, mTOR/PI3Kinh). Window trials in patients with TNBC have shown kinome reprogramming in response to MEKinh, suggesting understanding kinome resiliency is necessary for rational design of effective combination therapies to treat cancer in individual patients. Citation Format: Gary Johnson. Breast cancer subtypes have selective kinome reprogramming in response to kinase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY10-02. doi:10.1158/1538-7445.AM2013-SY10-02

Metastatic progression of tumors is the major cause of death in patients with triple negative breast cancer (TNBC), the most aggressive subtype of breast cancer. However, since metastasis is a multi-step process, unraveling its complexity is a major challenge. One effective way of tackling this question is to study natural blockers of the metastatic process, metastasis suppressors, and identify the mechanisms by which they regulate metastasis. Raf kinase inhibitory protein (RKIP), a protein that regulates kinase activity, is a suppressor of TNBC metastasis. Although RKIP inhibits the activity of key kinases such as Raf-1, GRK2, NIK/IKK in cultured cells, the kinase targets of RKIP in tumors are not known. To address this question, we used a mass spectrometry approach involving inhibitor-conjugated beads to identify kinases that are down-regulated by RKIP in human TNBC xenograft tumors. Our results identified novel targets of RKIP whose kinase activity is either down-regulated or up-regulated by RKIP. We used bioinformatics analysis to build RKIP-regulated signaling networks based upon RKIP-induced changes in kinase activity and related gene expression. Elucidating RKIP function at a systems level reveals the interplay between key metastatic signaling cascades, particularly in relation to cell migration and invasion, and can potentially identify novel anti-metastatic target combinations in TNBCs. Citation Format: Ali E. Yesilkanal, Casey Frankenberger, Daniel C. Rabe, Gary L. Johnson, Marsha Rosner. Elucidating metastatic signaling networks in TNBC by investigating RKIP-regulated kinome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1620.

Crosstalk between the tumor and surrounding microenvironment has emerged as an important regulator of tumor growth, metastasis and drug response. Patient-derived breast xenografts (PDXs) closely mimic the tumor microenvironment including the tumor architecture and interactions among cancer cells and stromal cells. PDXs provide a unique opportunity to study tumor-stroma interactions and the regulation of protein expression in the tumor microenvironment since species-specific amino acid sequences of the tumor (human) can be distinguished from the stroma (mouse) by LC-MS. However, quantitative proteomics workflows usually do not report species-specific peptides. We therefore developed a proteomics workflow based on 10-plex isobaric tagging to quantitatively profile the protein expression of PDXs and their associated microenvironment. Three biological replicates of seven breast cancer PDXs, representing three breast cancer subtypes, were profiled. Selecting only gene- and species-specific peptides for quantification of protein expression, we identified 8,113 human proteins (4,867 genes) and 2,251 mouse proteins (1,763 genes). Surprisingly, hierarchical clustering by mouse protein expression tightly clustered 4 of the 7 PDXs, with each of the 3 biological replicates next to one another. Notably, the 4 tightly clustered PDXs were from tumors with claudin-low, Her2-E and luminal B subtypes, whereas the biological replicates of three basal subtypes in the dataset were not tightly clustered. Gene set enrichment analysis of the stromal protein expression revealed upregulation of MTORC1 signaling, EMT, and interferon gamma response signaling with false discovery rates below 5%. We further investigated expression of signaling proteins in the tumor microenvironment by enriching active kinases with multiplexed kinase inhibitor beads. 152 mouse kinases were identified in the tumor microenvironment many tumor-specific differences in kinase levels. Taken together, our results imply that individual patient-derived breast tumors can actively and consistently orchestrate unique alterations in the proteins expressed in their microenvironment. Furthermore, we demonstrate the utility of our proteomic analysis workflow to delineate tumor-stroma signaling networks in PDXs. Citation Format: Xuya Wang, Petra Erdmann-Gilmore, Rosa Viner, Matthew Meyer, Tim Stuhlmiller, Sherri Davies, Shunqiang Li, Qiang Zhang, Arshag Mooradian, Kuan-lin Huang, Ryan Bomgarden, Li Ding, Matthew Ellis, John Rogers, Gary Johnson, Reid Townsend, David Fenyo, Jason M. Held. The proteomic landscape of patient-derived breast cancer xenografts reveals tumor-specific differences in the breast tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-267.

Introduction: Nearly half of metastatic triple negative breast cancer (TNBC) patients develop brain metastases (BMs) and face a poor prognosis. There are no FDA-approved systemic therapies to treat TNBC BM, due in part to the blood-brain barrier. BCBMs exhibit both activation of the PI3K pathway and AURKA amplification/overexpression relative to primary breast cancers. In this study, we evaluate the efficacy of brain-penetrant, clinically-available inhibitors of PI3K and AURKA in TNBC cell lines that are capable of growing in the mouse brain. Methods: In vitro characterization of the pan-PI3K inhibitor BKM120 and AURKA inhibitor MLN8237 were conducted in 2 human-derived TNBC cell lines, SUM149 and (MDA-MB-)231Br. A siRNA screen (720 kinase genes) was used to identify synthetic enhancers of lethality with BKM120 treatment. To assess the efficacy of these drugs, the IC50s of BKM120 and MLN8237 and synergy of the combination were determined. To compare the effects of BKM120 and/or MLN8237 treatment on cell cycle progression, FACS analysis was conducted at 24, 48, and 72 hours in parent cells and in cells continuously cultured in MLN8237-treated media for 12 weeks. Results: The screen confirmed that combined PI3K and AURKA inhibition synthetically enhanced lethality in SUM149 and 231Br cells. SUM149 and 231Br cells and two additional TNBC cell lines (MDA-MB-468 and MDA-MB-436) exhibited similar IC50s (1.3-21 μM) to BKM120. However, there was a >2.5 fold range (26.5-69 μM) in IC50s for MLN8237, with the greatest potency in the 231Br line. Concurrent treatment with BKM120+MLN8237 was synergistic or additive in 231Brs at most doses, whereas the combination was additive to antagonistic in SUM149s. Pretreatment with MLN8237 prior to concurrent BKM120+MLN8237 improved synergy in SUM149s, while BKM120 pretreatment improved synergy in 231Brs. FACS analysis of BKM120 in the SUM149 and 231Br cells induced a slight G1 arrest from 24 to 72 hours, while MLN8237 initially induced a G2 arrest at 24 hours, polyploidy at 48 hours, and a mixed polypoid/G2 arrested population at 72 hours. These effects were more pronounced in the 231Brs than the SUM149s. Combined BKM120+MLN8237 in both cell lines yielded results similar to MLN8237 alone. Cells continuously exposed to increasing MLN8237 concentrations from 50 nM to 300 nM for 12 weeks were resistant to MLN8237-induced cell cycle changes as compared to passage-matched controls. Conclusions: Combined PI3K+AURKA inhibition using brain-penetrant compounds is a promising strategy for a patient population with few options. In vivo studies evaluating the efficacy of BKM120+MLN8237 in intracranial TNBC mouse models to provide the translational foundation for future clinical studies are warranted. Citation Format: Amanda E.D. Van Swearingen, Maria J. Sambade, Shivani Sud, Brian Golitz, Gary L. Johnson, Carey K. Anders. Combined PI3K and AURKA inhibition are efficacious in triple-negative breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3867.

Although the number of therapies entering clinical trials continues to increase, the success rate for targeted therapy studies is quite low due, in part, to a lack of relevant preclinical models. The use of targeted therapy has become a popular method for cancer treatment against mutant BRAF melanoma, as studies show that clinical use of selective inhibitors against MEK and BRAF extend life expectancy. However, melanoma becomes refractory to treatment. Since the tumor and stromal factors that contribute to drug resistance remain poorly understood, our ability to treat patients with advanced disease remains limited. Current genetically engineered murine (GEM) models in melanoma research have enhanced our understanding of tumor biology and therapeutic response, but fail to mimic tumor progression in clinical settings as most lack spatial and temporal control of endogenous tumor initiation. To better mimic human disease, we have developed a novel tamoxifen application method that reproducibly induces local tumor formation on the mouse ear, and we have incorporated a tdTomato fluorescent reporter allele (tdTomatoLSL) into an existing tamoxifen-inducible GEM model of BRAFV600E/PTEN-null melanoma. The tdTomato allele serves as a visual marker of Cre recombination in endogenous melanocytes and allows disease progression to be followed through macroscopic and multiphoton intravital imaging. To investigate therapeutic effects at the cell level, we have performed imaging studies of primary tumors before and after treatment with a selective MEK1/2 inhibitor (MEKi), Trametinib, which is a FDA approved therapy for malignant melanoma. Our melanoma induction method enables longitudinal studies of drug response that ultimately give rise to fully resistant tumors (up to 90 days on MEKi). Our studies show that this treatment causes a striking morphological change of the tumor cells as early as 3 days post-treatment. After several weeks of continuous treatment, the remaining tumor cells are highly spatially correlated with bundled collagen structures detected by second harmonic signal, suggesting that cellular milieu strongly influences drug response. Interestingly, our current studies with MEKi suggest that association of tumor cells with collagen fibrils initially provides a protective effect against this drug, however once true resistance emerges, this dependency on extracellular matrix in the tumor stroma diminishes. Furthermore, in parallel with our imaging studies, we have performed molecular analysis of tumor response using Mib/MS and mRNA-seq. By coupling our imaging studies with transcriptome and kinome reprogramming analysis, we hope to identify properties that promote resistance. Using these novel approaches, we have developed a model that enables direct observation of endogenous tumor development, plasticity, and resistance to targeted therapy at the cell level in situ for the first time. Citation Format: Hailey E. Brighton, Steven Angus, Tao Bo, David Darr, Norman E. Sharpless, Gary L. Johnson, James E. Bear. Intravital imaging of endogenous BRAFV600E melanoma reveals plasticity of tumor response and resistance to trametinib over time. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4382.

s: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Metastatic progression of tumors is the major cause of death in patients with triple negative breast cancer (TNBC), the most aggressive subtype of breast cancer. However, since metastasis is a multi-step process, unraveling its complexity is a major challenge. One effective way of tackling this question is to study natural blockers of the metastatic process, metastasis suppressors, and identify the mechanisms by which they regulate metastasis. Raf kinase inhibitory protein (RKIP), a protein that regulates kinase activity, is a suppressor of TNBC metastasis. Although RKIP inhibits the activity of key kinases such as Raf-1, GRK2, NIK/IKK in cultured cells, the kinase targets of RKIP in tumors are not known. To address this question, we used a mass spectrometry approach involving inhibitor-conjugated beads to identify kinases that are down-regulated by RKIP in human TNBC xenograft tumors. Our results identified novel targets of RKIP whose kinase activity is either down-regulated or up-regulated by RKIP. We used bioinformatics analysis to build RKIP-regulated signaling networks based upon RKIP-induced changes in kinase activity and related gene expression. Elucidating RKIP function at a systems level reveals the interplay between key metastatic signaling cascades, particularly in relation to cell migration and invasion, and can potentially identify novel anti-metastatic target combinations in TNBCs. Citation Format: Ali E. Yesilkanal, Casey Frankenberger, Daniel C. Rabe, Gary L. Johnson, Marsha R. Rosner. Elucidating metastatic signaling networks in TNBC by investigating RKIP-regulated kinome. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B23.

Introduction: Nearly half of metastatic triple negative breast cancer (TNBC) patients develop brain metastases (BM) and face a poor prognosis. The blood-brain barrier (BBB) prevents many treatments from reaching intracranial tumors, and there are no FDA-approved systemic therapies to treat TNBC BM. In this study, we evaluated the efficacy of BBB-permeable, clinically-available inhibitors of MEK and identified rational co-target pathways in preclinical models of intracranial (IC) TNBC. Methods: In vitro IC50s, synergy, and siRNA screens (700 kinase genes) were conducted in 4 human-derived TNBC lines (SUM149, MDA-MB-468, MDA-MB-436, MDA-MB-231Br). We evaluated the efficacy of the MEK1/2 inhibitor AZD6244 (AZD), the pan-PI3K inhibitor BKM120 (BKM), and the PDGFR inhibitor Pazopanib (Pazo) in IC TNBC mouse models. Tumor burden was monitored via bioluminescence, and IC tumors were frozen for gene expression analyses using custom human 4×44K Agilent microarrays or of kinome activity profiles using multiplex kinase inhibitor beads and mass spectrometry. To determine drivers of AZD sensitivity, DNA copy number data (Broad CCLE) was analyzed using SWITCHplus to identify copy number alterations that differ between sensitive (n = 8) vs. resistant (n = 12) TNBC lines based on their IC50s (Sanger Cancerxgene). Results: In vitro, SUM149 and 231Br TNBC cells exhibited lower (40 uM). Several genes synthetically enhanced lethality in SUM149 and 231Br cells: PI3K genes and PDGFRα/β with AZD, and MAPK/MAP2K/MAP3K genes with BKM, suggesting MEK+PI3K and MEK+PDGFR inhibition as rational combinations. AZD plus BKM or Pazo were synergistic in vitro in sensitive cell lines. In vivo, AZD reduced tumor burden and improved survival in the SUM149 (72 vs. 45 days in controls, p < 0.005) and 231Br (37 vs. 30 days, p < 0.02) models, with no benefit in the other two models. Single agent BKM or Pazo resulted in little to no improvement in any model. However, in AZD-sensitive models, combined AZD+BKM inhibition increased survival (SUM149: 87 vs 45 days, p < 0.0001; MDA-MB-231Br: 52 vs 30 days, p < 0.001) as did AZD+Pazo (SUM149: 88 vs. 35 days, p < 0.0001). Several DNA segments were significantly altered in sensitive vs. resistant TNBC cell lines. Notably, MEK-pathway genes were lost in the resistant lines. Ongoing work will complete characterization of therapies in all models in vitro and in vivo and will compare genetic, transcriptional, and kinome activity alterations. Conclusions: TNBC models exhibit different innate sensitivities to the BBB-permeable MEK inhibitor AZD6244. In sensitive models, AZD improves survival and reduces intracranial tumor burden, and rational combined inhibition of PI3K or PDGFR further increases survival. Identification of predictive biomarkers will enable translation of our results to biomarker-driven clinical trials for patients with TNBC BM. Citation Format: Amanda E.D. Van Swearingen, Marni B. Siegel, Maria J. Sambade, Shivani Sud, Samantha M. Miller, Grace Silva, Ryan E. Bash, Charlene M. Santos, David B. Darr, Brian Golitz, Joel S. Parker, C. Ryan Miller, Gary L. Johnson, Carey K. Anders. Combination therapy with MEK inhibition is efficacious in intracranial triple negative breast cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2579. doi:10.1158/1538-7445.AM2015-2579

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with very low survival rates. Metastatic progression of tumors is the major cause of death in TNBC patients. Metastatic spread of tumor cells is generally associated with resistance to therapy and poor prognosis. Therefore, there is a clinical need for understanding the molecular and cellular basis of metastasis and developing effective therapies against metastatic progression. However, since metastasis is a multi-step process, unraveling its complexity is a major challenge. One effective way of tackling this question is to study natural blockers of the metastatic process, metastasis suppressors, and identify the mechanisms by which they regulate metastasis. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor in TNBC, which acts as a homeostatic factor that regulates kinase activity. RKIP blocks invasion in vitro and experimental metastasis in vivo. Therefore, RKIP constitutes a powerful tool for studying metastatic signaling pathways in cancer cells. RKIP is known to regulate the activity of certain kinases such as Raf-1, GRK2, NIK, and GSK3β, affecting major pathways such as MAPK, NFκB, and WNT. However, our preliminary data suggests that RKIP affects a much larger part of the TNBC kinome. Using proteomics approaches, we have identified putative novel kinase targets of RKIP, and used bioinformatics analysis to build RKIP-regulated signaling networks. Elucidating RKIP's function at a systems level will reveal the interplay between key metastatic signaling cascades and potentially identify novel anti-metastatic target combinations in TNBCs. Citation Format: Ali E. Yesilkanal, Casey Frankenberger, Daniel Rabe, Gary L. Johnson, Marsha R. Rosner. Regulation of TNBC kinome by the metastasis suppressor Raf kinase inhibitory protein (RKIP). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3251. doi:10.1158/1538-7445.AM2015-3251

KRAS mutational activation plays a critical role in tumorigenesis, but exactly which downstream KRAS effector pathway is critical for this role remains less clear. One of the most studied downstream pathways is the phosphatidylinositol-3-kinase (PI3K) pathway which mediates cellular metabolism, growth, and survival. We have previously shown using validated pancreatic ductal adenocarcinoma (PDAC) patient derived xenograft (PDX) mouse models that treatment with BKM120 (a pan-class 1 PI3K inhibitor, currently in Phase I/II clinical trials) resulted in tumor growth inhibition (p = 0.017) but not regression. In this study, we evaluated possible mechanisms of resistance to BKM120 therapy. We developed a novel multiplex inhibitor bead/mass spectrometry (MIB/MS) assay to measure the activation state of the kinome. We have recently shown that kinome response to kinase inhibitor therapy (i.e. kinome reprogramming) is a potential mechanism of resistance in triple negative breast cancer in response to MEK inhibition and in drug-resistant leukemia in response to imatinib. We hypothesized that kinome reprogramming may play a role in resistance to PI3K inhibition in PDAC and used MIB/MS to identify second targets that may be used in combination with PI3K inhibitors. We found kinome reprogramming in response to PI3K inhibition through both previously known as well as less studied pathways. For instance, we found MEK1 and MEK2 activation in response to BKM120 treatment. MEK activation has previously been implicated in resistance to PI3K inhibition and combined MEK and PI3K inhibition has been shown to be synergistic in PDAC. However, our results suggest that resistance to PI3K may be mediated through many more pathways than MEK alone. We found ErbB1, ErbB2 and ErbB3 activation in response to BKM120 treatment in both cell lines and PDX tumors. ErbB2 and ErbB3 activation has previously been noted in response to PI3K inhibition in breast cancer. However, no studies have evaluated this combination in PDAC. Thus we evaluated the effect of combined PI3K and pan-ErbB inhibition in a panel of 10 PDAC cell lines using BKM120 and dacomitinib (a pan-ErbB inhibitor currently in Phase III clinical trials). Combined treatment with BKM120 and dacomitinib inhibited proliferation in 10 of 10 PDAC cell lines evaluated. This combination showed impressive synergy across all cell lines with a mean combination index of 0.24 (0.00245 – 0.49). MIB/MS is a powerful unbiased approach to identify second targets for combination therapy. We identified both known and novel kinase pathways that may mediate resistance to PI3K inhibition in PDAC. Our results suggest that pan-ErbB inhibition may be a promising second target in combination with PI3K inhibition in PDAC. Combination studies in PDX models are ongoing. Pan-ErbB and PI3K inhibition in PDAC may be more effective than either single agent alone and should be considered in clinical trials. Citation Format: Christopher J. Tignanelli, Jeran Stratford, Richard Moffitt, Rachel Reuther, Gary L. Johnson, Jen Jen Yeh. Multiplexed kinase inhibitor beads identify multiple pathways of resistance to PI3K inhibition facilitating the rational selection of novel combination therapies in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B71.

Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype which lacks the classical clinical biomarkers for hormone receptors (HR) and HER2. Nearly half of metastatic TNBC patients develop brain metastases and these patients face a poor prognosis. Most new effective treatments for breast cancer target hyperactivated HER2 and HR pathways, which TNBC tumors lack. The blood-brain barrier (BBB) also prevents many treatments from reaching the brain and any developing tumors therein. There is currently no FDA-approved systemic chemotherapy for the treatment of TNBC brain metastases. Tissue-based studies show activation of the PI3K pathway in breast cancer brain metastases and in vivo models of extracranial TNBC are sensitive to PI3K and MEK inhibitors. BBB-permeable inhibitors targeting the PI3K and MEK pathways are in clinical development. In this study, we evaluated the efficacy of two such inhibitors, the pan-PI3K inhibitor BKM120 and the MEK1/2 inhibitor AZD6244, in preclinical models of intracranial (IC) TNBC. Methods: The efficacy of PI3K and MEK inhibition by BKM120 and AZD6244, respectively, alone and in combination was determined in orthotopic mouse models of basal-like TNBC through IC implantation of SUM149 or MDA-MB-468 cell lines. Drugs were administered at the maximum tolerated doses via chow: 30 mg/kg BKM, 37 mg/kg AZD, or 25 mg/kg BKM + 18 mg/kg AZD. Tumor burden was monitored via weekly bioluminescence imaging, and brain tumors were frozen at sacrifice for gene expression analyses. To explore potential resistance mechanisms and inform rational combination therapies, in vitro IC50s, combination synergy determinations, and single-agent synthetic lethal siRNA screens were conducted. Results: In the SUM149 model, median survival was 45 days (control), 53.5 days (BKM), 53.5 days (BKM+AZD), and 76 days (AZD). AZD alone and with BKM reduced tumor burden via bioluminescence imaging. In vitro, 3-day dose response curves showed that BKM and AZD were similarly potent but BKM was more effective (IC50; maximal % cell reduction: BKM: 1.3 uM, >90%; AZD: 816 nM, >50%). Several genes were identified as synthetically lethal “hits” in an initial kinome screen by two-class SAM. AURKA was a hit with BKM120, while BRAF and several PI3K, AMPK, and CDK genes were synthetically lethal with AZD6244. Ongoing work will include survival studies in the 468 model and gene expression changes in IC tumors in response to therapy. Conclusions: BKM120 and AZD6244 both improved survival in an IC TNBC SUM149 mouse model, with single agent AZD6244 being most efficacious. The siRNA screens indicate that combined treatment with BKM120 and AZD6244 should be synthetically lethal, suggesting that combination therapy may have underperformed due to toxicity. Ongoing in vitro and in vivo studies (including dosing schedules) will further characterize the effects of these drugs in intracranial TNBC. Citation Format: Amanda E.D. Van Swearingen, Marni B. Siegel, Ryan Bash, Brian Golitz, Charlene Santos, David Darr, Joel Parker, Gary L. Johnson, C. Ryan Miller, Carey K. Anders. PI3K and MEK inhibition in intracranial triple negative breast cancer: Efficacy of BKM120 and AZD6244 in preclinical mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5449A. doi:10.1158/1538-7445.AM2014-5449A

Early clinical studies have shown that concurrent administration of BRAF and MEK inhibitors, dabrafenib and trametinib, is more active in patients with BRAFV600E/K melanoma than either single agent alone. Even though the combination of BRAF/MEK inhibitors is initially highly effective in treating melanoma in a subset of patients, progression to resistance ultimately occurs–commonly via reactivation of the BRAF/MEK/ERK pathway. Therefore, therapeutic strategies to counter primary resistance and prevent the emergence of secondary resistance are needed. Following pharmacological or progressive genetic perturbations, dynamic and system-wide adaptive changes (“reprogramming”) in the expression and activity of multiple kinases (collectively termed the kinome) clearly occur in tumor cells. Rich networks of serine/threonine and tyrosine kinases, many of which are understudied, are integrated into the signaling systems controlled by oncogenes known to drive melanoma. Thus, a comprehensive understanding of the kinome at baseline, during the adaptive response to kinase inhibitor treatment, and in the context of acquired resistance is critical. We use Multiplexed Inhibitor Beads (MIBs), mixtures of covalently immobilized, linker-adapted kinase inhibitors coupled with mass spectrometry (MS), to assess changes in activation of kinases in untreated samples, during acute inhibition of BRAF and/or MEK and in the context of acquired resistance. Our enhanced MIB/MS technology allows us to study over 75-80% of the expressed kinome (and numerous lipid and metabolic kinases) simultaneously using lysates from cell lines, genetically engineered mouse model (GEMM) tumors, patient-derived xenografts or human melanoma. We have identified several kinases (e.g. PDGFRβ, DDR1, MAP3K1) that are activated upon acute treatment with dabrafenib, trametinib, or dabrafenib/trametinib as well as in drug-resistant human melanoma cells and BRAFV600E/PTEN-/- GEMM-derived tumors and cell lines. Thus, studies using MIB/MS to measure dynamic kinome responses have identified kinases that have not been previously shown to be involved in the adaptive response to BRAF and/or MEK inhibition. We are currently targeting kinases involved in adaptive kiome reprogramming, pharmacologically or by RNAi, to determine whether their activation leads to vulnerability. This work will lead to the identification of novel and rationally predicted therapies for patients with advanced melanoma. Citation Format: Steven P. Angus, Timothy J. Stuhlmiller, Rachel Reuther, Trang T. Pham, Deborah A. Granger, David B. Darr, Jamie L. Jordan, Stergios J. Moschos, Gary L. Johnson, Norman E. Sharpless. Defining the adaptive kinome response to BRAF and MEK inhibition in melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4761. doi:10.1158/1538-7445.AM2014-4761