Adam Marcus - US grants

The Cell Imaging and Microscopy Core (CIMC) provides cellular imaging technologies and services to enhance the programmatic research initiatives of the Winship Cancer Institute (WCI). It is centrally located in the WCI and houses state-of-the-art technologies including point scanning confocal, live cell confocal, and spectral confocal imaging, as well as epifluorescence microscopy, image processing, and image analysis. To ensure that that these technologies are properly utilized, CIMC personnel perform comprehensive training sessions and provide consultation on experimental setup, execution, and analysis. Moreover, CIMC personnel provide daily user assistance and perform routine maintenance on all equipment to ensure optimal image acquisition and analysis. The CIMC has also developed an educational web site that provides online scheduling, recent upgrades, and describes the technologies and applications available. The CIMC is a WCI Core, and the WCI is responsible for oversight, operations, budget, compliance, user fee collection, and employee relations. The Core director makes all day-to-day decisions with input from CIMC personnel, users, and internal advisory boards. CIMC services have impacted numerous programmatic initiatives that include biomarker discovery and development, cancer nanotechnology, tumor diagnostics, drug delivery, molecular pharmacology, and cell biology. Furthermore, the CIMC participates in two institution-wide grants and collaborates with several WCI cores and investigators. As a result, the CIMC has users from all WCI programs, and WCI members comprise 78% of total CIMC usage. The CIMC has focused on two areas of technology development[unreadable] cancer nanotechnology and more recently, in vivo (small animal) cellular imaging. These new technologies strengthen the scientific repertoire of the CIMC and ultimately enhance the basic, translational, and clinical initiatives of the WCI.

DESCRIPTION (provided by applicant): Though clinicians can predict which patients are at risk for developing metastases, traditional therapies prove ineffective and metastatic disease is the primary cause of cancer patient death. We propose a novel anti- metastatic strategy to prevent metastases from occurring by perturbing a unique cell migration target-the vimentin cytoskeleton. Vimentin overexpression is found in nearly all invasive cancers in both basic and pre- clinical models, and is used as a clinical marker of cancer invasion. Functional studies show that vimentin is essential for cell migration but not viability, and overexpression leads to more aggressive and motile cancer cells. Here we present data showing that Withania somnifera root extracts (WRE) target vimentin to inhibit cancer cell motility and invasion while having negligible effects on cell viability at low doses. We test the hypothesis that WRE is an anti-metastatic and anti-invasive complementary alternative medicine (CAM) that disrupts vimentin function with limited toxicity. We propose comprehensive analysis of the anti-invasive and anti-metastatic efficacy of WRE in cell lines and a pre-clinical metastatic mouse model. We will employ cutting- edge live cell imaging to dissect the mechanism of how WRE inhibits vimentin function and cancer cell migration, as well as determine if WRE inhibits endothelial cell motility resulting in anti-angiogenic activity. Since all data show that vimentin disruption does not impact cell viability, this cell invasion inhibitor is aligned with the goals of CAM and chemoprevention. Ultimately, we envision that WRE can be a vimentin-targeting chemopreventative in high-risk metastatic patients. PUBLIC HEALTH RELEVANCE: Metastatic disease is the major cause of death in almost all cancer types;however, most treatments target the primary tumor and not the metastases. Here we take a chemopreventative approach to prevent cancer invasion and metastasis from ever occurring. We propose studies that investigate the efficacy of our novel compound, its mechanism of action, and pre-clinical activity.

DESCRIPTION (provided by applicant): Lung cancer is the most lethal malignant cancer worldwide and results in over 150,000 deaths per year in the United States. In particular, non-small cell lung cancer (NSCLC), which accounts for nearly 80% of all lung cancers, has a 5-year survival rate ranging from only 15-25%. Numerous reports show that the intermediate filament protein vimentin is overexpressed in invasive human tumors but is nearly undetectable in non- invasive, stationary tumors. In NSCLC, vimentin expression correlates with poor survival, increased metastatic disease, and poor differentiation. Nevertheless, the mechanistic role of vimentin in NSCLC is unexplored. Here we show that the STRAD1-LKB1 lung cancer tumor suppressor pathway, which is mutated in 30% of NSCLC patients and is the 3rd highest mutated pathway in NSCLC, regulates vimentin function during lung cancer motility. Thus, we link vimentin to a robust NSCLC tumor suppressor pathway. We will test the central hypothesis that during lung cancer invasion, vimentin is overseen by STRAD1-LKB1 and participates in a positive feedback loop that maintains directionality persistence. Our objectives are to determine how STRAD1-LKB1 oversees vimentin function, how vimentin goes on to regulate NSCLC motility, and the molecular and clinical consequences of vimentin expression in NSCLC patients. Importantly, we have published that STRAD1-LKB1 interact with the canonical cell polarity and motility proteins cdc42-PAK1. We build upon this data to determine whether STRAD1-LKB1 regulate vimentin through cdc42-PAK1. Moreover, we propose that vimentin then goes on to regulate NSCLC directionality persistence through a positive feedback loop containing cdc42, and the cdc42 guanine exchange factor (GEF) VAV2. We take an innovative and comprehensive mechanistic approach by combining state-of-the-art cell and molecular biology, in vivo xenograft models, and patient tissue-based approaches to fully translate these findings. By understanding how STRAD1-LKB1 regulates vimentin and how vimentin expression contributes to NSCLC metastasis, we can impact our understanding of the biology of LKB1 mutant (~50,000 patients) and vimentin overexpressing NSCLC patients. Thus, this proposal can develop a new paradigm for vimentin function in NSCLC and present vimentin as a major player in the regulation of lung cancer metastatic invasion.

PROJECT SUMMARY (See instructions): The Cell Imaging and Microscopy Shared Resource (CIM) provides cellular imaging technologies and services to enhance the programmatic research objectives of the Winship Cancer Institute (Winship). It is centrally located in Winship and houses state-of-the-art technologies including point scanning confocal, live cell confocal, spectral confocal imaging, epifluorescence microscopy, and image processing and analysis. CIM services have supported numerous Winship programmatic initiatives that include cancer cell biology, biomarker development, cancer nanotechnology, tumor diagnostics, drug delivery, and molecular pharmacology. These interactions have resulted in high Impact publications, team science grants, and inter- and intra-programmatic collaborations. Winship members comprise 88% of total CIM usage, with overall usage increasing from 1533 hours in calendar year 2008 to 4332 hours in 2010. CIM personnel provide consultation on experimental setup, execution, and analysis to ensure optimal image acquisition and data analysis. This includes comprehensive training sessions and dally user assistance to ensure that technologies are properly utilized. Alt user image data are archived nightly onto NAS enterprise level RAID protected servers to provide secure data backup. The CIM is a Winship shared resource, and Winship is responsible for oversight, operations, budget, compliance, user fee collection, and employee relations. The CIM Director makes all day-to-day decisions with input from CIM personnel, users, an internal advisory board, and executive oversight committee. The primary objectives of the CIM over the next two years are to expand small animal fluorescence imaging resources, implement a new assay development service, and continue to expand our IT infrastructure. These new technologies along with existing CIM services will enhance Winship's basic, translational, and clinical initiatives of Winship.

The onset of "big data" has led to an increased appreciation of the value of large data sets in both industrial and academic settings. Typically, the analysis of such data sets begins by modeling the data as a high dimensional system, and so an understanding of the behavior of such systems (and techniques for dealing with them) has become equally important. This award supports a mathematical investigation of the behavior of such systems using techniques introduced by the researcher in previous work with collaborators. These techniques have led to the resolution of a number of open problems across various mathematical fields, including the Kadison-Singer problem and the existence of Ramanujan graphs of all sizes and degrees. This project aims to further develop substantial connections with research across numerous fields of science, including convex optimization, real algebraic geometry, functional analysis, combinatorics, and probability. The award also supports plans to focus interdisciplinary activities of particular benefit to students associated with the project.

On a technical level, the project focuses on two areas of research: (A) to develop a theory of ''finite free probability,'' a collection of ideas lying in the intersection of random matrix theory, convex optimization, real algebraic geometry, and polynomial geometry, and (B) to generalize the previously mentioned techniques so as to widen their potential application. This includes extending the ideas to bivariate polynomials as well as a non-Hermitian setting. In the direction of (B), the project aims to address some of the obstacles that hinder the application of the current techniques (for example, the need for real rootedness of associated polynomials) by investigating analogous behaviors in more general settings. This would be a necessary part of extending (A) to non-Hermitian settings, for example, as such objects no longer satisfy the conditions necessary to apply the current techniques. Extensions in this direction would also open the possibility of application to new areas such as quantitative geometry.

? DESCRIPTION (provided by applicant): The 3rd most commonly mutated gene in lung adenocarcinoma is the kinase LKB1 (STK11), where ~25,000 patients have this mutation. Patients that are co-mutated for LKB1 and KRAS have significantly shorter overall survival compared to either mutation alone and have no effective targeted therapies. In a genetically engineered Kras mouse model, co-mutation of Lkb1 is sufficient to promote metastasis of lung adenocarcinoma, and primary tumors and metastases show aberrant activation of both EMT and adhesion signaling. In agreement with these data, we have published three reports that LKB1 regulates lung cancer motility and when present, restrict the activity of a key adhesion signaling molecule, focal adhesion kinase (FAK). Moreover, our preliminary data show that Lkb1-deficient tumor cells invade as a collective pack in vivo, and in doing so, remodel the collagen ECM of the tumor microenvironment, a phenomenon that is dependent on the activity of FAK in vitro. These metastatic tumors from the mouse model show aberrant FAK activation within collective invasion packs of the primary tumors. Therefore, we hypothesize that LKB1-mutant cells use EMT to initially invade through the basement membrane, which is followed by defective FAK-based adhesion signaling that allows cells to navigate the collagen microenvironment during metastasis. To test this, we will take a multi-model approach that combines the power of Drosophila and mouse genetics, and our large clinically annotated human lung adenocarcinoma tumor bank to i) test the mechanism and signaling pathway used by Lkb1-mutant cells to traverse the basement membrane, ii) determine whether altered adhesion signaling drives in vivo cell escape through collagen remodeling, and iii) determine whether LKB1-mutant human lung adenocarcinoma exhibits altered adhesion signaling that correlates with collagen remodeling and predicts poor outcome. We propose that this comprehensive approach can overcome the shortcomings of traditional in vitro systems and address our goal of defining the biological consequences of LKB1 mutations in lung cancer patients.

? DESCRIPTION (provided by applicant): Lung cancer is the leading cause of cancer death in the United States, with the majority of patients having non-small cell lung cancer (NSCLC). The 3rd most commonly mutated gene in NSCLC adenocarcinoma is the serine/threonine kinase LKB1, where ~25,000 patients have this mutation. Targeted therapies for LKB1 mutant patients do not exist thereby creating a significant unmet clinical need. In a transgenic genetically engineered mouse model (GEMM), the combination of Lkb1 and Kras mutations promote metastasis and create treatment-refractory tumors compared to Kras-only mice. Genomic and proteomic profiling show that these mice have increased expression of pro-metastatic pathways, notably the focal adhesion kinase (FAK). We published that LKB1 is indeed a FAK repressor in vitro, such that when LKB1 is absent, phosphorylated FAK (pFAK397) is hyperactivated. Consistent with this work, we now present preliminary in vivo data that pFAK397 is activated at the invasive front of high-grade metastatic tumors within the KrasG12D Lkb1fl/fl GEMM. This prompted us to test the intriguing possibility that pharmacologic FAK inhibition in this GEMM would repress metastasis due to pFAK397 inhibition. Our preliminary data show that treatment with a FAK inhibitor suppresses the rate of metastasis, suggesting that targeting FAK can be a viable anti-metastatic strategy for treating LKB1 mutant tumors. Similarly, FAK inhibitor treatment using in vitro Lkb1 null spheroids also potently inhibits invasion and suppresses pFAK397 activation. Based upon these preliminary and published data, we hypothesize that LKB1 inactivation in NSCLC represents a unique acquired tumor vulnerability that can be targeted pharmacologically with a FAK inhibitor. To test this hypothesis the objectives of this proposal are i) to determine if pharmacologic FAK inhibition can specifically suppress the metastasis of Lkb1-mutant lung tumors in vivo and ii) define which clinically observed LKB1 mutations cause pFAK397 activation and create a tumor vulnerability targetable by pharmacologic FAK inhibition. These objectives will be met by performing pre-clinical mouse trials in a unique and clinically-relevant GEMM to examine FAK inhibition as a strategy for inhibiting metastatic disease in Lkb1 mutant tumors. In addition, we will implement a novel LKB1 sequencing pipeline in patient samples from our clinically-annotated tumor bank, and determine if specific LKB1 mutations correlate with pFAK activation and clinical outcome. We have assembled a team of interdisciplinary investigators to facilitate the translational and multi-pronged nature of these studies. Since FAK inhibitors are in Phase I and II trials, we propose that the data from this proposal will provide a rational foundation to develop a future trial with FAK inhibitor specifically targeting patients with LKB1 loss-of-function mutations.

? DESCRIPTION (provided by applicant): The NIH Science Education Partnership Award (SEPA) program of Emory University endeavors to use an over-arching theme of Citizen Science principles to meet the goals of this proposal are to: 1) develop an innovative curriculum based on citizen science and experiential learning to evaluate the efficacy of informal science education in after-school settings; 2) promote biomedical scientific careers in under-represented groups targeting females for Girls for Science summer research experiences; 3) train teachers in Title I schools to implement this citizen science based curriculum; and 4) disseminate the citizen science principles through outreach. This novel, experiential science and engineering program, termed Experiential Citizen Science Training for the Next Generation (ExCiTNG), encompasses community-identified topics reflecting NIH research priorities. The curriculum is mapped to Next Generation Science Standards. A comprehensive evaluation plan accompanies each program component, composed of short- and/or longer-term outcome measures. We will use our existing outreach program (Students for Science) along with scientific community partnerships (Atlanta Science Festival) to implement key aspects of the program throughout the state of Georgia. These efforts will be overseen by a central Steering Committee composed of leadership of the Community Education Research Program of the Emory/Morehouse/Georgia Institute of Technology Atlanta Clinical Translational Science Institute (NIH CTSA), the Principal Investigators, representatives of each program component, and an independent K-12 STEM evaluator from the Georgia Department of Education. The Community Advisory Board, including educators, parents, and community members, will help guide the program's implementation and monitor progress. A committee of NIH-funded investigators, representing multiple NIH institutes along with experienced science writers, will lead the effort for dissemination and assure that on-going and new NIH research priorities are integrated into the program's curriculum over time.

? DESCRIPTION (provided by applicant): Genomic analysis has revealed significant molecular heterogeneity within single tumors. This heterogeneity remains a major obstacle to understanding the biological drivers of tumor progression. To that end, we have developed a new imaging-based technique to obtain genomic profiles of any cell or population by precisely selecting and extracting living cells of interest from their native environment. These purified and extracted cells can then be subjected to genomic analysis, or amplified and cultured for molecular studies. We have termed this image-guided genomics technique, spatiotemporal genomic analysis (SAGA). Due to our long-standing interest in cancer invasion and metastasis, we have used SAGA to probe cancer invasion by focusing on collective tumor cell invasion, which is major mode of metastasis in murine models and patients. We have used SAGA to: 1) perform the first genomic analysis directly comparing the genomic expression profile of purified highly invasive leader cells of the collective invasion pack, to follower cells, which stream behin the leader cells. 2) select, amplify, and maintain the first purified leader cell lines and followe cell lines, which now give us virtually unlimited quantities of previously rare invading cell types Our genomics data show that leader cells have distinct expression profiles with overexpression of key invasion-related genes compared to follower cells. Since we can keep these rare cell types in culture, and they maintain their respective phenotype over time, we show that leader cells invade aggressively yet proliferate poorly, whereas followers show the converse by invading poorly and proliferating rapidly. Importantly, mixing these two cell types leads to a synergy where leader cells reprise their roles and actively seek out follower cells to promote collective invasion; follower cells, in turn, promote effective leader cell proliferation. These observations lead us to tumor cell specialization within the collective invasion pack results in increased metastatic success due to cell-cell cooperativity. hypothesize that If true, it suggests that collectively invading cells represent a specialized labor force that are specifically primed t promote invasion To test this, we will determine if cell specialization within the invasion unit dictates invasive potential, primary tumor formation, and metastasis to a secondary site by moving to in vivo assays with our unique cell lines. Then we will probe the molecular basis of leader cell invasive behavior by leveraging our current genomic data set and determine which genes drive leader and follower cell behavior and communication during collective cell invasion. Taken together, these approaches provide a unique opportunity to understand the mechanistic underpinnings of rare cell types and heterogeneity within the collective invasion pack and proliferation.

INTEGRATED CELLULAR IMAGING SHARED RESOURCE ? PROJECT SUMMARY/ABSTRACT The Integrated Cellular Imaging Shared Resource (Imaging SR; Category 1.33) provides state-of-the-art light microscopy and image analysis services to Winship investigators. Sixteen imaging systems are available at four sites established to serve Winship members throughout Emory's main campus. Imaging SR services support Winship research efforts in biomarker discovery and development, cancer cell biology, tumor diagnostics, drug delivery, and molecular pharmacology. Imaging SR is utilized by members of all four Winship research programs, with 35-40 Winship members' laboratories using Imaging SR each year during the current CCSG funding cycle. This utilization has supported work published in high impact journals and supported preliminary data leading to grants for investigators in all four programs. Winship members have access to novel technologies, including state-of-the-art multiphoton microscopes, super-resolution imaging, and live-cell imaging. Winship members receive services at a subsidized rate for Imaging SR usage. Winship supports dedicated Imaging SR staff (one faculty and three staff). To ensure these technologies are properly utilized, Imaging SR personnel provide comprehensive training sessions and consultation on experimental setup, execution, and analysis. Imaging SR personnel provide daily user assistance and perform maintenance on all equipment to ensure optimal image acquisition and analysis. The Imaging SR director has managed the Winship imaging shared resource facilities since 2005 and is a tenured associate professor with an NCI-funded laboratory. The mean total hourly usage by Winship members during the current funding cycle has been 3,486 hours/year, representing 52% of overall Imaging SR utilization by Winship members.

? DESCRIPTION (provided by applicant): The 3rd most commonly mutated gene in lung adenocarcinoma is the kinase LKB1 (STK11), where ~25,000 patients have this mutation. Patients that are co-mutated for LKB1 and KRAS have significantly shorter overall survival compared to either mutation alone and have no effective targeted therapies. In a genetically engineered Kras mouse model, co-mutation of Lkb1 is sufficient to promote metastasis of lung adenocarcinoma, and primary tumors and metastases show aberrant activation of both EMT and adhesion signaling. In agreement with these data, we have published three reports that LKB1 regulates lung cancer motility and when present, restrict the activity of a key adhesion signaling molecule, focal adhesion kinase (FAK). Moreover, our preliminary data show that Lkb1-deficient tumor cells invade as a collective pack in vivo, and in doing so, remodel the collagen ECM of the tumor microenvironment, a phenomenon that is dependent on the activity of FAK in vitro. These metastatic tumors from the mouse model show aberrant FAK activation within collective invasion packs of the primary tumors. Therefore, we hypothesize that LKB1-mutant cells use EMT to initially invade through the basement membrane, which is followed by defective FAK-based adhesion signaling that allows cells to navigate the collagen microenvironment during metastasis. To test this, we will take a multi-model approach that combines the power of Drosophila and mouse genetics, and our large clinically annotated human lung adenocarcinoma tumor bank to i) test the mechanism and signaling pathway used by Lkb1-mutant cells to traverse the basement membrane, ii) determine whether altered adhesion signaling drives in vivo cell escape through collagen remodeling, and iii) determine whether LKB1-mutant human lung adenocarcinoma exhibits altered adhesion signaling that correlates with collagen remodeling and predicts poor outcome. We propose that this comprehensive approach can overcome the shortcomings of traditional in vitro systems and address our goal of defining the biological consequences of LKB1 mutations in lung cancer patients.

PROJECT SUMMARY/ABSTRACT Emory University?s Lung Cancer SPORE aims to promote the advancement of early career lung cancer investigators through the conduct of a Career Enhancement Program (CEP). The goals of the proposed Lung Cancer SPORE CEP are to provide training and guidance for academic physician-scientists, clinician- investigators, and laboratory-based scientists who wish to dedicate their efforts to translational research in the areas of diagnosis, imaging, prevention, treatment, and improvement in quality of life in lung cancer, and to enhance diversity among the lung cancer research community. The SPORE CEP will build on the strong commitment of the Emory Winship Cancer Institute and the associated School of Medicine Departments to the career development of junior investigators, which has included 18 career development program awardees as part of Emory?s previous head and neck cancer SPORE and an ongoing T32 training grant in molecular oncology. The proposed CEP aims to provide an environment that enables talented early career investigators to engage in a 2-year mentored research program and to facilitate their success and their academic career development in terms of achieving independent investigator status. Specific aims for the CEP are as follows: Aim 1: To attract and mentor a talented group of early career investigators towards a career in translational lung cancer research; Aim 2: To engage biologists, scientists and physicians present at Emory University and local partner research institutions in the state of Georgia in translational lung cancer research; Aim 3: To enhance diversity among the lung cancer research community by encouraging minority individuals, women, and individuals with disabilities to apply for CEP support; and Aim 4: To foster and guide emerging strategies for the study of the biology, prevention and long term treatment of lung cancer. On average, 2 projects will be funded annually. Additional support for the CEP has been secured from matching funds from the Winship Cancer Institute, Emory University School of Medicine and the Woodruff Health Sciences Center.

Abstract Collective invasion is a major mode of metastasis observed in patients across most solid tumor types. How the collective invasion pack operates, communicates, and navigates as a single cohesive unit remains unclear. To address this, we published on an image-guided genomics platform to isolate any living cell(s) within a collective invasion pack, and expand the population for genomic and molecular analysis, a technique we termed Spatiotemporal Cellular & Genomic Analysis (SaGA). We used SaGA to deconstruct the collective invasion pack and dissect the molecular profiles of leader and follower cells invading as a hierarchical cohesive unit. To generate the collective invasion pack, leader and follower cells undergo a VEGF/Notch-based angiogenic mimicry program that promotes cell:cell cooperation and invasion that is similar, but not identical to angiogenesis. VEGF secreted by invasive leaders recruits proliferative followers into the collective pack; once the pack is formed, leader and follower cells undergo a Notch1-Dll4 cell patterning program that includes the Dll4 antagonist, Jagged-1 (Jag1). Based upon our published and preliminary data, we hypothesize that cooperative signaling among contiguous cells via Notch1 and its ligands are required to form the spatially dependent signaling events within the invasion pack. We propose that this fosters cell:cell cooperation and leads to increased metastatic efficiency. To test this, in Aim 1 we will define how atypical angiogenic mimicry via Notch1/Jag1/Dll4 signaling operates to spatially regulate cooperation and invasion. This would be a significant step forward in understanding how this pathway operates to maintain the collective invasion pack, drive metastasis, and facilitate ECM remodeling. In Aim 2, we use Jag1 as a lung cancer leader cell biomarker to isolate the first patient leader cells and probe atypical angiogenic mimicry. This allows us to define the metastatic potential and translational impact of this rare yet invasive population in lung cancer patients. Throughout, we leverage unique resources developed here including SaGA-derived cell lines, the first set of early and late-stage invading lung patient-derived organoids, ex vivo imaging, and a rare set of lung primary tumors with paired metastatic brain tissue. We speculate that these data will provide mechanistic insight into the atypical angiogenic mimicry program and translational value towards understanding lung cancer patient leader cell biology.

Project Summary Collective invasion is a major mode of metastasis observed in patients across most solid tumor types. How the collective invasion pack operates, communicates, and navigates as a single cohesive unit remains unclear. To address this, we published on an image-guided genomics platform to isolate any living cell(s) within a collective invasion pack, and expand the population for genomic and molecular analysis, a technique termed Spatiotemporal Cellular & Genomic Analysis (SaGA). We used SaGA to dissect the molecular, epigenetic, and genomic profiles of leader and follower cells invading as a hierarchical cohesive unit. To determine how epigenetic reprogramming drives this phenotypic heterogeneity, we deconstructed the collective invasion pack using SaGA, then integrated genome-wide promoter methylation and transcriptome data to define differentially methylated regions within the leader and follower phenotypes. We observe global epigenomic re-wiring in leader cells supporting an epigenetic basis for the phenotypic heterogeneity within the collective invasion pack. We then identified Myo10 (myosinX) as a top differentially methylated and expressed gene, where the leader cell promoter is hypomethylated, and leaders in several lung cancer lines overexpress Myo10. Myo10 is a canonical modulator of filopodia elongation and we show it drives filopodia elongation, collective invasion, leader cell-driven fibronectin micropatterning (fibrillogenesis), and is transcriptionally activated by Jag1/Notch. We will use this information to test a mechanistic model with the overarching hypothesis that Myo10 activation via promoter hypomethylation in leader cells drives filopodia-based micropatterning of fibronectin to create a leader cell-driven collective invasion path. We propose that this leads to an invasive advantage for lung cancer cells resulting in metastatic disease. In Aim 1 we test the model that Myo10 hypomethylation in leaders allows for Jag1/Notch1- driven transcriptional activation, driving filopodia elongation, and fibronectin micropatterning. In Aim 2 we test how this collective invasion pathway impacts metastasis using in vivo metastasis models and the first patient- derived leader cells. Throughout, we leverage unique resources developed here including SaGA-derived cell lines, ex vivo imaging, and patient-derived lung cancer leader cells. We speculate that these data will provide mechanistic insight into collective invasion and translational value towards understanding lung cancer patient leader cell biology.

Project Summary Most studies investigate whole cancer cell populations and rarely address how single cells or sub-populations cooperate to promote their survival and spread. This is especially true in the context of metabolism, where tumors harbor distinct sub-populations of glycolytic and oxidative cells forged in part by microenvironmental pressures. How this metabolic heterogeneity drives tumor invasion and metastasis is an unexplored area and requires approaches that can isolate these specific cancer cell sub-populations. This multi-PI application in lung adenocarcinoma attempts to address this by determining how metabolically heterogeneous cancer cell sub- populations function and cooperate to drive invasion and metastasis. We build upon our published image-guided genomics technology (SaGA) to extract living and phenotypically defined cell sub-populations within collectively invading packs. We used SaGA to deconstruct the lung cancer collective invasion pack, which is comprised of hierarchical groups of invasive leader and proliferative follower cells invading as a cohesive unit. Our published and preliminary data show that follower cells consume twice as much glucose as leaders, and rely on glycolysis and the oxidative pentose phosphate pathway (PPP) to maintain their proliferative state. By simply disrupting the glucose transporter, GLUT1, followers become invasive and take on a leader-like phenotype. In contrast, leaders rely on oxidative phosphorylation (OXPHOS) via pyruvate dehydrogenase (PDH) activity to drive invasion, where disrupting PDH creates a more follower-like phenotype. These data lead to our overarching hypothesis that metabolic heterogeneity sustained by differential GLUT1 and PDH-driven metabolism facilitates lung cancer metastasis by maintaining distinct phenotypes in the collective invasion pack. We propose that this metabolic heterogeneity warrants a co-targeting therapeutic approach that disrupts both metabolic populations to inhibit metastasis. Thus, the objective of this proposal is to 1) elucidate the molecular basis and mechanistic underpinnings of how metabolic heterogeneity drives collective invasion and 2) test if co-targeting different metabolic sub-populations limits metastasis. We propose that these studies will directly impact our understanding of how metabolic heterogeneity sustains cooperativity to promote collective invasion/metastasis.

PROJECT SUMMARY/ABSTRACT - Cancer Clinical Investigator Team Leadership Award (CCITLA) Dr. Eaton is an academic radiation oncologist with a research focus on therapeutic clinical trial development involving novel approaches of radiotherapy for pediatric and adult brain tumor patients. Her goal is to lead institutional investigator-initiated and NCI-funded cooperative group clinical trials that will further advance the fight against cancer. Dr. Eaton plays a critical role in Winship Cancer Institute's clinical trial program and is extensively involved in NCI funded clinical trials. She is as an active member in multiple national committees of NRG Oncology and the Children's Oncology Group (COG), and the Pediatric Brain Tumor Consortium (PBTC). She is the institutional principal investigator of two NCI-funded clinical trials through NRG Oncology and five investigator initiated clinical trials at Winship, and contributes as an active co-investigator on numerous others. As a radiation oncology leader among the Winship brain tumor pediatric oncology programs, Dr. Eaton is an important contributor to patient enrolment to NCTN trials at Winship, playing a major role in the Winship Lead Academic Participating Site (LAPS award) award from the NCI. She is an integral member of Winship's Clinical and translational Research Committee (CTRC) and serves as the Pediatric Medical Director of the Emory Proton Therapy Center (EPTC), which opened in 2018 as the first proton center in the state of Georgia. In this role, Dr. Eaton led the EPTC Imaging and Radiation Oncology Core (IROC) credentialing process necessary for participation in NCTN clinical trials, critical to the successful opening of 12 NCI funded clinical trials at EPTC and supporting NCTN clinical trial accrual with the adoption of this advanced technology in the state of Georgia. Winship Cancer Institute of Emory University fully supports Dr. Eaton in her career goals to advance as a national leader in clinical trials for brain tumor patients and pediatric malignancies, and in her ongoing efforts to enhance the clinical trial program at Winship. The CCITLA award will support protected time for Dr. Eaton to focus on key initiatives that will develop infrastructure and process improvements to expand clinical trials and increase trial accrual at Winship, and further develop her own promising protocol concepts at the national level. The CCITLA funding mechanism will undoubtedly help foster academic growth and lead to further leadership roles within the Department of Radiation Oncology, Winship Cancer Institute, Emory University, as well as the NCI Trial Networks.