Pre-clinical cancer studies have been limited by the availability of patient cell models that accurately represent the diversity of patient populations. The utility of these studies is further impacted by the lack of normal tissue from the same patient. Furthermore, clinical evaluation of targeted therapeutics, like tarceva (erlotinib), would benefit from patient models expressing the target for robust characterization of drug response. Moreover, individual patient responses to the same anti-cancer compound cannot be assessed with high precision unless healthy controls are used from the same tumor donor. Here, we present six breast cancer patient-derived tumor models, accompanied by cancer (tumor)-associated fibroblasts (CAFs) and mesenchymal stem cells (MSCs). These paired and matched sample cohorts were studied in the context of spheroid models of the metastatic breast cancer niche. We show that these models allow an increased understanding of chemotherapy modulation by non-cancerous cells, which are present within the tumor but not targeted by the current chemotherapies or other anti-tumor approaches. We used a three-drug set to create a viability profile for each patient-associated tumor sample, which in turn can be used to aid clinical decision-making and increase the personalization of each treatment for an individual. Further, our approach shows different genomic and proteomic profiles for the co-culture models when compared between the tumor 3D and healthy controls. Citation Format: Liam Deems, Dmitry Shvartsman, Maria Ivanova, Cheryl Murphy, David Deems. Enabling therapeutic decisions for a breast cancer patient cohort using matched diseased and normal tissue in tumor organoids [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 P5-06-15.

Abstract Cancer research relies on a plethora of disease-relevant tumor models, but patient specific models are still being developed. In this study, we show the utility of patient-specific pancreatic, ovarian and breast cancer models in the context of the metastatic niche, composed of co-cultured mesenchymal stem cells (MSC) in three-dimensional tumor spheroids. Additionally, different ratios of MSC's were seeded with the cancer spheroids to interrogate the effect of MSC's on chemotherapeutic drug response in defined media conditions. The patient-specific and treatment-naive cancer models represent a diverse range of disease type, progression grade, and genetic profiles. These models were co-cultured with MSC's sourced from both bone marrow and adipose tissue. These co-culture models were tested with a panel of common, broad-range chemotherapeutic agents as well as several disease-specific drugs. The resulting drug responses show that the presence of MSC's within the context of the metastatic niche causes attenuation towards a higher chemotherapeutic drug resistance. The tumor models exhibit a varied chemotherapeutic response when MSC's are seeded in 50:1, 5:1, or 1:1 ratio with cancer cells, suggesting that even a small presence of MSC's may have a significant effect on the drug resistance of metastatic tumor spheroids. While this MSC-induced drug response attenuation is observed across the broad spectrum of patient profiles and disease types, the strength of the effect is not homogenous. The observed variability in the strength of the effect was impacted by the disease type of the cancer cells, the source of the MSC's, and the specific drug used in the metastatic tumor model. We propose that using these reproducible and easily scaled models of metastatic tumors to find effective chemotherapeutic drugs could increase our understanding of the tumor microenvironment, the onset of metastasis, and the process of cancer grafting into other tissues. Discovering effective and targeted therapies utilizing a more advanced model of the metastatic tumor niche could increase the success rate of an eventual clinical trial. Citation Format: Liam Deems, Amit Shahar, Amy Pepicelli, Cheryl Murphy, Daniel Solomon, David Deems, Dmitry Shvartsman. Modeling the metastatic niche interactions between patient tumor and mesenchymal cells to identify drivers of chemotherapy drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3164.

Abstract Creating patient-specific models with a high level of characterization and functionality in tumoroid modeling enables a better understanding of the disease drivers in metastatic cancers. It presents the opportunity for personalized treatment with novel therapeutic reagents in underrepresented cancers such as pancreatic ductal adenocarcinoma. Using a defined and treatment-naive patient-specific model cohort of nine patients diagnosed with pancreatic adenocarcinoma, we aimed to represent the diversity of progression grade and assay profiles. These models were tested with a panel of common, broad-range chemotherapeutic agents and characterized for genetic, proteomic, and tumoroid drug response profiles. A detailed analysis of gene expression values from the original tissue specimen, compared with cancer cultures in hypoxic, normoxic planar, and spheroid (3D) culture conditions, enabled the identification of C-MET as a critical gene that shows upregulation closely resembling the expression profile observed in the original patient biopsy and was modulated by tissue culture format. Once this potential target was identified, the pancreatic models were treated with five C-MET-targeting chemotherapeutic agents with different modes of action. Proto-oncogene receptor C-MET (HGF receptor) is a potential therapeutic target in many metastatic cancers due to its integral role in angiogenesis, proliferation, and cancer cell survival¹. We propose that these reproducible and easily scaled tumoroid models can be used to find new therapeutic targets in-vitro and provide the example of C-MET in our pancreatic cancer patient cohort. Moreover, only 3D hypoxic models closely resembled original patient tissues in their genomic expression profiles. Using these model formats, we distinguished differences between various C-MET targeting compounds and their ability to induce cell death, which correlated with their mode of action on C-MET neutralization. Identifying these targets in 3D produces new and potentially more effective drug targets where they may not have been observed solely in planar conditions with legacy cell lines. Our models of metastatic pancreatic cancer tumoroids can be readily utilized to benefit researchers and clinicians seeking new targets for patient treatment. Citation Format: Liam Deems, Maria Ivanova, Cheryl Murphy, Amit Shahar, David Deems, Dmitry Shvartsman. Identification of C-MET receptor as a therapeutic target in patient-specific tumoroid models of metastatic pancreatic adenocarcinoma allows identification of a new mode of action for its inhibitors [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-074.

Abstract Non-small cell lung cancer (NSCLC) is a prevalent and deadly disease because of high incidence and relapse rates. One hypothesis for the high relapse rate is the existence of cancer stem cells (CSCs), a rare subpopulation of cells within these tumors, that are resistant to therapy and thought to be responsible for local and distal recurrence. CSCs are also able to self-renew and differentiate into multiple cell types forming the mass of new tumors. These differentiated progeny that constitute the bulk of the tumor are more sensitive to radiation and chemotherapeutic agents, express tissue of origin markers, and have an intrinsically limited lifespan. While most solid tumors have these properties, the degree of heterogeneity from patient to patient in the multi-lineage potential of the CSC population is not known. In particular, NSCLC CSC populations from primary patient tumors have not been well studied. The goal of this study was to characterize newly derived NSCLC CSC lines to determine the degree of heterogeneity between patient samples, and to provide a starting point for the discovery of novel differentiation therapeutic agents to target CSCs. We isolated six CSC lines from primary patient NSCLC tumors using an unbiased culture-based enrichment method, rather than a biased marker-based approach. Each of these six CSC lines was tumorigenic in immunodeficient mice at low cell numbers and the resulting tumors recapitulated the original tumor histology. All-trans retinoic acid (ATRA), a well-known differentiation agent for the treatment of acute promyelocytic leukemia (APL), maintains the normal growth and differentiation of human bronchial epithelial cells in culture. Each of the six CSC lines was treated with ATRA for two weeks to induce differentiation and then assessed for transcript and protein levels of candidate CSC and differentiation markers. Pre-treatment, the CSC lines all expressed CD44. Post-ATRA treatment, the majority of the CSC lines expressed Mucin-2, a marker of Goblet cell lineage. In addition, each cell line started to show evidence of the very early stages of differentiation into other lineages including Clara Cells, Neuroendocrine Cells, and Alveolar Type I and Type II cells. In one CSC line, ATRA treatment both in vitro and in vivo delayed tumor cell growth, induced the expression of Mucin-2 and decreased the expression of Nestin, a cancer stem cell marker. To further characterize CSC differentiation potential, we screened a pilot set of small molecules in two independent CSC lines to identify compounds that induce terminal differentiation, using Mucin-2 expression as readout. Surprisingly, despite the fact that these two CSC lines were from different histological subtypes (adenocarcinoma and squamous cell carcinoma) and displayed different transcriptional profiles post-differentiation, there was a good correlation between hit sets in the screens. Taken together, these data suggest that while the CSC of origin in tumors differs between patient samples in profile and function, there is promise to identify differentiation agents that are broadly active in different NSCLC subtypes. Further screening is currently being performed to identify these novel differentiation agents for therapeutic use. Citation Format: Dina Shlyakhter, Diane Boucher, Yong Gu, Amy B. Hall, Elaine Krueger, Anna Lindquist, Cheryl Murphy, Yuxin Wang, Mark Wood, Brenda Eustace. Differentiation screen identifies small molecules that target histologically divergent subtypes of patient-derived lung cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B24.

Proficient repair of DNA damage is important for cancer cell survival and is a leading cause for the poor response many patients experience when treated with DNA-damaging drugs or ionizing radiation. The protein kinase ataxia telangiectasia mutated and Rad3 related (ATR) regulates an important DNA damage response pathway that is most commonly activated by replication stress (RS). RS arises during S-phase when the cell9s DNA replication machinery attempts to copy through an unresolved damage lesion. Such events are common after cells are treated with DNA-damaging agents. Unresolved RS often leads to double strand breaks, which in turn may cause DNA mutations, chromosomal rearrangements or cell death. Pre-clinical data suggests a reliance on ATR for survival is a common feature in cancer cells. This may occur when there are defects in other DNA damage repair pathways or high levels of background RS. VX-970 is the first potent (Ki VX-970 is currently in Phase 1 clinical studies as monotherapy and in combination with gemcitabine, cisplatin and carboplatin. Note: This abstract was not presented at the meeting. Citation Format: John Pollard, Philip Reaper, Julie Jones, Christopher Barnes, Scott Gladwell, Stuart Hughes, Adele Peak, Hakim Djeha, Amy Hall, David Newsome, Yuxin Wang, Diane Boucher, Brenda Eustace, Yong Gu, Brian Hare, Mac Johnson, Sean Milton, Cheryl Murphy, Darin Takemoto, Crystal Tolman, Mark Wood, Brinley Furey, Marina Penney, Howard Li, Christopher Defranco, Mohammed Asmal, Scott Fields. VX-970, the first-in-class inhibitor of the DNA damage repair enzyme ATR. [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 1644. doi:10.1158/1538-7445.AM2015-1644

Abstract DNA damaging agents have been the cornerstone of cancer therapy for decades yet they provide only modest benefit for most patients. For example, standard of care for patients with non-small cell lung cancer (NSCLC) is dominated by the use of platinating drugs and ionizing radiation (IR), however outcome remains very poor with 5-year survival rates of <15% for patients that present with advanced disease. Such poor responses to DNA damaging treatment reflects, in part, the efficient repair of DNA damage via a complex signaling and repair network known as the DNA damage response (DDR). The DDR detects double strand breaks and replication stress, the most lethal forms of DNA damage, and acts to enforce checkpoints to halt cell cycle progression, and to stimulate repair. Key regulators of the DDR are the phosphoinositol 3-kinase-like serine/threonine protein kinase (PIKK) family members ATR and ATM. Recent pre-clinical data has suggested that a reliance on ATR for survival from DNA damage may be a common feature of cancer. This can arise either as a consequence of high replicative stress, for example from expression of certain oncogenes, from a hypoxic microenvironment, or from defects elsewhere in DNA damage surveillance and repair pathways. Most notably inhibition of ATR has been shown to be synthetic lethal with loss of the ATM-p53 pathway. In NSCLC defective ATM signaling, from loss of ATM expression or from defects in p53 has been reported in about 50% of tumors. Here we describe the comprehensive in vitro and in vivo profile for VE-822 a novel highly potent and selective inhibitor of ATR. VE-822 potently inhibits ATR in biochemical assays with Ki <0.3nM and in cell assays with IC50 of 20nM. Against a large panel of NSCLC lines, low concentrations of VE-822 sensitized many lines to the cytotoxic effects of multiple DNA damaging agents; for example >90% of lines showed >3-fold shifts in IC50 for cisplatin in the presence of VE-822, with ~50% of lines showing >10-fold increases in cisplatin cytotoxicity. In contrast normal cells tolerate inhibition of ATR. In a panel of mouse xenograft models, derived from various primary human NSCLC tumor tissues, oral or IV administration of VE-822 strongly sensitized tumors to cisplatin treatment. In many cases, combinations including VE-822 led to tumor regression or extensive tumor growth delay. Inhibition of ATR activity and accumulation of DNA damage by VE-822 was observed coincident with efficacy. When administered alone or in combination with cisplatin VE-822 was well tolerated in mice at doses that block ATR activity. These data support the potential for ATR inhibitors to substantially increase the efficacy of standard-of-care agents in diseases such as NSCLC. Citation Format: Diane Boucher, Peter Charlton, Jean-Damien Charrier, Brinley Furey, Yong Gu, Amy Hall, Brian Hare, Howard Li, Sean Milton, Cheryl Murphy, Philip Reaper, Darin Takemoto, Taturo Udagawa, Yuxin Wang, Mark Wood, John Pollard. Comprehensive preclinical evaluation of VE-822, the first ATR-targeted drug candidate: a novel approach to transforming the efficacy of DNA damaging agents. [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 LB-299. doi:10.1158/1538-7445.AM2013-LB-299

Abstract DNA damaging agents have been the cornerstone of solid cancer therapy for decades yet they provide only modest benefit for patients with many tumor types. This reflects, in part, the efficient repair of DNA damage via a complex signaling and repair network known as the DNA damage response (DDR). Key regulators of the DDR are the phosphoinositol 3-kinase-like serine/threonine protein kinase (PIKK) family members ATR, ATM and DNA-PK. The DDR acts to detect DNA lesions, enforce checkpoints to halt cell cycle progression, and stimulate repair. Recent data have shown that elements of the DDR are commonly defective in cancer cells. It is widely believed that these cells become dependent on the remaining DDR pathways for survival from DNA damage. Inhibitors have been reported for a number of DDR enzymes, including ATM, DNA-PK, CHK1 and PARP, however there are no reports of drug-like ATR inhibitors. Here we disclose the in vitro characterization of a potent and highly selective ATR inhibitor (VE-821). This compound selectively blocks ATR signaling in cells (IC50 = 0.7 µM), but has little impact on ATM or DNA-PK signaling (IC50 >10 µM). Treatment with 10 µM VE-821 for 144 h causes little cell death in normal cell lines (5-11 %) but markedly higher death in cancer cell lines (28-46 %). VE-821 also dramatically sensitizes many cancer cells to multiple classes of genotoxic agents including antimetabolites, topoisomerase inhibitors and crosslinking agents; with over 10-fold increases genotoxic potency observed in some cases. In a panel of 36 lung cancer cell lines, VE-821 sensitized the cytotoxic effect of cisplatin to a far greater magnitude and over a broader subset of these lines than potent inhibitors of ATM, Chk1, or PARP. In over half of these cell lines, the IC50 of cisplatin was reduced by greater than 5 fold upon the addition of VE-821. We show that a basis for the cancer-selective effects of VE-821 is a synthetic lethal interaction between loss of ATM signaling (a frequent event in cancer resulting from loss of function of proteins such as ATM or p53) and ATR inhibition when cells encounter DNA damage. In keeping with this, ATR inhibition does not sensitize normal cells (with functional ATM) to the cytotoxic effects of genotoxic therapy. In this case a compensatory DDR is activated that is associated with marked activation of ATM, which in turn leads to reversible checkpoint arrest and a strong survival response. These studies show for the first time that a selective ATR inhibitor can preferentially sensitize cancer cells to genotoxic drugs by exploiting a synthetic lethal interaction between ATM and ATR signaling. This underpins the broad potential of ATR inhibition as a highly promising new strategy to improve the efficacy of genotoxic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5491. doi:10.1158/1538-7445.AM2011-5491

PURPOSE: Pulmonary function abnormalities have been detected in many obese children enrolled exercise programs. Such abnormalities may affect the exercise tolerance and fitness outcomes of such programs. METHODS: 88 obese children were enrolled in the Downstart Pediatric Healthy Lifestyle program from 2004-2006 at SUNY Downstate. Downstart offers behavior modification thru nutrition education and exercise for inner city children and adolescents. All subjects were obese according to Am. Academy of Pediatrics guidelines. The length of the program was 12 weeks. The pre-participation evaluation included pulmonary function (PFT)s as per ATS recommendations. BMI, fat percentage, and fitness capacity were measured before and after the 12 week program. 35 individuals had non-normal PFT's with a median age of 11.75 and an avg. BMI of 33.9. 53 had normal PFT's with a median age of 12.5 and an avg. BMI of 34.47. Fitness testing was based on the guidelines of the President's Council on Physical Fitness and Sports. It included push ups, sit ups, squats, a step test, and sit & reach test of flexibility. Subjects were placed into one of two categories, based on PFT's. RESULTS: 39.77% of participants had respiratory dysfunction (obstructive or restrictive or both) confirmed by PFT. Results showed that children with normal lung function had greater improvements in fitness when compared with those with abnormal PFT's in almost all categories. Those with non-normal lung function had statistically significant improvement in 1 category (push-ups) while those with normal lung function had improvement in 3 categories (BMI, push-ups, and squats).TABLECONCLUSION: Reduced lung function affects improvement in fitness for obese children.

Marked energy expenditure, independent of energy balance, could change whole-body protein turnover. The aims of the present study were to determine the effect of participation in a 6-day, 10-stage cycling stage tour on whole-body protein turnover in elite male cyclists, and to determine whether energy and protein turnover are related to fatigue and over-reaching. 13C-leucine was used to determine 18-h whole-body protein turnover in cyclists both before and immediately after a cycling stage race. The 18-h period included two feeding periods to simulate a normal evening meal and a normal breakfast meal, and two fasted periods including overnight. Blood was drawn for the determination of plasma cortisol and serum ferritin on days 2, 4, and 6 and a Profile of Mood States questionnaire was administered on alternate days during the tour event for determination of markers of over-reaching. Mean leucine rate of appearance was unchanged from pre- to post-tour during both fed and fasted conditions and mean energy balance was maintained. Serum ferritin concentration declined and plasma cortisol concentration remained stable over the 6 days. Markers of overtraining were evident in one athlete who pulled out of the tour event due to fatigue on the second to last day. Our main finding was that high energy output over a 6-day period did not significantly change protein turnover during fed, fasted or overnight conditions.

Research measuring whole-body protein turnover (WBPT) after both exercise and nutrition has generally focused on resistance exercise; however, there is a paucity of data regarding the effect of postaerobic exercise nutrition, especially in older adults. It is not known if postexercise protein feeding has a beneficial effect on protein turnover after low- to moderate-intensity exercise. We investigated whether consuming protein plus carbohydrate (PRO) immediately after an acute bout of aerobic exercise has an additive effect over carbohydrate alone (CHO) on WBPT in older individuals. Twelve healthy older adults (age, 59 ± 4 years) were studied on 2 separate occasions after 1 h of exercise at approximately 50% of maximal rate of oxygen uptake, followed by 4 h of recovery. Immediately following exercise, subjects ingested a CHO (60 g) or an isocaloric PRO beverage (40 g carbohydrate, 20 g whey protein). Whole-body protein metabolism was determined using [1-13C]leucine infusion (60 mg prime; 75 mgh(-1) continuous), and sampling blood and expired breath. Rates of whole-body leucine appearance and oxidation, and nonoxidative leucine disposal during the third and fourth hours of postexercise recovery were higher in the PRO group (2.51 ± 0.55, 0.78 ± 0.37, and 1.71 ± 0.44 micromol kg(-1)·min(-1), respectively) than in the CHO group (1.81 ± 0.27, 0.33 ± 0.14, and 1.47 ± 0.25 micromol kg(-1)·min-1, respectively; p = 0.001). Our results indicate that consumption of a PRO beverage after aerobic exercise increased WBPT to a greater extent than a CHO beverage.

PURPOSE: Weight management programs promoting changes in behavior through nutrition and exercise education are advocated to reverse the prevalence of obesity. The optimum length of such programs, however, remains unclear. Shorter programs may be economically attractive, but may not be sufficient to establish changes in behavior. In this study we compared 8 and 12 week exercise programs for obese youth. METHODS: The 151 subjects were enrolled for 8 weeks (28 M, 35 F)or 12 weeks (35 M, 53F) median age 12.25 and 12.33 yrs and mean BMI of 34.29 and 34.07 respectively, in the Downstart Pediatric Healthy Lifestyle program from 2004-2006. Downstart offered by Downstate Medical Center in Brooklyn offers an intense program of behavior modification using nutrition and exercise intervention. All participants were inner city children and adolescents who were obese according to American Academy of Pediatrics guidelines. The BMI and fat % of all participants were measured before and after the program. Similarly, fitness testing was done before and at end of the program. The fitness testing was based on the guidelines established by the President's Council on Physical Fitness and Sports and included push ups, sit ups, squats, a 2 minute step test, and a sit & reach test of flexibility. Subjects were placed into two groups based on program length. RESULTS: In all but one category (fat %) individuals in the 12 week program outperformed their peers in the 8 week program. The difference between the two groups was statistically significant in three categories (step-test, push-ups and squats). The height increased by 0.78cm and 1.39 cm respectively in the 8 and 12 week programs.TABLECONCLUSION: 12 week exercise program led to statistically significant improvement on fitness tests such as the step-test, push-ups, squats when compared to the shorter 8 week program.

The rate of whole body protein synthesis in healthy adults is depressed during aerobic exercise and stimulated afterwards. Whether this situation persists in older individuals is currently unclear. Postprandially, in both young and old, the rate of whole body protein synthesis is elevated above baseline measures. Research measuring whole body protein turnover after both exercise and nutrition has generally focused on resistance exercise. There is a paucity of data regarding the effect of post-aerobic exercise nutrition in older adults. PURPOSE: To investigate whether feeding protein immediately after an acute bout of aerobic exercise has an additive effect over carbohydrate alone on the rate of whole body protein synthesis in older individuals. METHODS: Twelve healthy older adults (59 ± 4 yrs) were studied on two different occasions at rest (2h), during 1h of exercise at 50% VO2max, and during 4h of subsequent recovery. Subjects ingested isocaloric carbohydrate-only (CHO; 60g) or carbohydrate-plus-protein beverages (PRO; 40g carbohydrate, 20 g whey protein) immediately following the cessation of exercise. Whole body protein metabolism was determined by ι-[1-13C]leucine infusion (60 mg prime; 75 mg/h continuous) with sampling of blood and expired breath. RESULTS: Rates of whole body leucine oxidation and non-oxidative leucine disposal during the third and fourth hours of post-exercise recovery were higher in PRO (0.75 ± 0.2 and 1.76 ±0.2 μmol/kg/min respectively) than CHO (0.34 ± 0.04 and 1.48 ± 0.1 μmol/kg/min respectively) (p = 0.001). CONCLUSIONS: The provision of protein in a post-aerobic exercise carbohydrate-containing beverage has a positive effect on whole body protein synthesis in older individuals. This is the first study to report the effects of post-aerobic exercise nutrition on whole body protein metabolism in older subjects. Supported by NZ HRC Grant 05/266 and FRST Bright Futures Doctoral Scholarship.

Protein phosphorylation is a primary form of information transfer in cell signaling pathways and plays a crucial role in regulating biological responses. Aberrant phosphorylation has been implicated in a number of diseases, and kinases and phosphatases, the cellular enzymes that control dynamic phosphorylation events, present attractive therapeutic targets. However, the innate complexity of signaling networks has presented many challenges to therapeutic target selection and successful drug development. Approaches in phosphoproteomics can contribute functional, systems‐level datasets across signaling networks that can provide insight into suitable drug targets, more broadly profile compound activities, and identify key biomarkers to assess clinical outcomes. Advances in MS‐based phosphoproteomics efforts now provide the ability to quantitate phosphorylation with throughput and sensitivity to sample a significant portion of the phosphoproteome in clinically relevant systems. This review will discuss recent work and examples of application data that demonstrate the utility of MS, with a particular focus on the use of quantitative phosphoproteomics and phosphotyrosine‐directed signaling analyses to provide robust measurement for functional biological interpretation of drug action on signaling and phenotypic outcomes.

3508 Tyrosine phosphorylation, a critical regulator of cellular signaling cascades, is dysregulated in various diseases, including cancer. Phosphorylation pathways overlap, intersect and feedback, creating complex signaling networks that requires a broad systems-level analysis to determine relevant phosphorylation patterns or “signatures” for defining disease models. An antibody array platform, with a peptide-based immunoassay design, was developed to detect 45 tyrosine phosphorylation sites on 34 different proteins simultaneously, in a single sample (Ti-Tyr™ Chip, Epitome Biosystems, Inc). As aberrant EGFR signaling has been implicated in various types of human cancers, we used this antibody array to identify and quantify time-dependent changes in phosphorylation downstream of the EGF receptor in HeLa cells, stimulated with EGF. Changes in site-specific phosphorylation of downstream targets were quantified for important regulators of migration (PLC gamma, FAK), cell proliferation (Her2, Shc, Crk, Erk) and receptor endocytosis /degradation (Cbl, Ack). Temporal and quantitative analysis demonstrated differences in EGF-induced p38 and Erk phosphorylation profiles. In addition, the cell surface receptor tyrosine kinases, EGFR and Her2, showed different temporal activation profiles suggestive of EGFR transactivation of Her2. Peptide-based immunoassays for tyrosine phosphorylation can be used for temporal and quantitative analysis of phosphorylation for profiling receptor tyrosine kinase signaling, at a site-specific level. Changes in tyrosine phosphorylation “signatures” can be used to monitor responses to pathway stimulation and therapeutic interventions.

Cellular homeostasis and responses to stimuli are mediated by complex signaling network events dominated by changes in protein phosphorylation states. Understanding information flow in the network is essential for correlating signaling changes to cell physiology. Tyrosine phosphorylation constitutes only a small portion of all protein phosphorylation, but its importance is manifested by the significant role it plays in diseases such as cancer. A peptide-based immunoassay microarray, designed to provide site specificity, quantification, broad coverage, and accessibility, is described that profiles 45 tyrosine phosphorylation sites across 34 proteins. Epidermal growth factor-stimulated A431 cells in the absence and presence of kinase inhibitors analyzed by microarrays showed biologically validated tyrosine phosphorylation changes and unanticipated activation of other targets. The approach is scalable for increasing the breadth of content as well as for interrogating other types of protein posttranslational modifications.

Broad screening of protein phosphorylation has wide‐ranging utility for understanding the impact of disease or drug treatment on cell signaling pathways. To address this need, we have developed an antibody array for measuring tyrosine signaling across 45 phosphotyrosine sites across multiple signaling pathways (Ti‐Tyr ™ Profiling Chip). The approach utilizes a novel strategy of measuring peptide fragments liberated by protease digestion of the sample prior to analysis, and allows for both multi‐site profiling and absolute quantification using synthetic peptide standards. The utility of the Ti‐Tyr™ chip is demonstrated by measuring specific modulation of pathway phosphorylation in stimulated cells in the presence and absence of distinct kinase inhibitors. Phosphorylation changes detected in chip targets correlate with known biological effects using comparable conditions. The use of peptide standards on the chip allows for an absolute quantitative assessment of the same stimulation conditions or inhibitor treatments across different cell lines, for example EGF stimulation in A431 and PC‐3 cancer lines. The Ti‐Tyr ™ Tyrosine Chip should improve the ability to understand the effects of cell stimuli, the underlying basis of disease and the impact of drug treatments across multiple cell pathways and cell types.

INTRODUCTION: Pediatric obesity continues to rise in the United States at alarming rates. Multi-disciplinary programs offering psychological counseling, nutrition education and exercise have been shown to have some favorable outcomes in increasing fitness and reducing weight. The optimum mode of exercise however, is unclear. A fundamental principal in exercise prescription is that the best mode of exercise is the one the subject enjoys performing the most. PURPOSE: To determine if the application of Shotokan Karate exercise can improve fitness in a program advocating the adoption of a healthy lifestyle. METHODS: Shotokan Karate is a very popular exercise modality, that has shown increases in physical fitness as well as in weight loss. (1) We introduced this popular Japanese discipline into the Downstart Healthy Lifestyle Program, an 8 week exercise, nutrition education and behavioral change program aimed at inner city children and adolescents in Brooklyn. The twice weekly sessions consisted of Kata training, emphasizing kicking, punching and blocking techniques with rhythmic breathing. Baseline physical fitness, flexibility and muscle strength was measured at the beginning and the end of the 8 week sessions, incorporating parameters established by the President's Council on Exercise RESULTS: Parameters were measured as a maximum completed in 60 seconds. Data is being reported as an average for the entire group. Push ups increased by 26%. Sit ups increased by 25%. Squats increased by 19%. Flexibility, as measured by sit and reach box, increased by 11%. Endurance, as measured with a two minute step test, increased by 14%. CONCLUSION: Our preliminary results indicate that the application of Shotokan Karate exercises in the Downstart Program may be a viable exercise modality for improving physical fitness. Further study is needed to determine if Shotokan Karate training is a greater motivating mode of exercise than conventional exercise programs currently available. References: 1-“Oxygen uptake, heart rate and blood lactate responses during and following Karate training” Imamura H, Yoshimura Y, Nishimura S, Nakazawa AT, Nishimura C, Shirota T. Department of Food and Nutrition, Nakamura Gakuen University, Fukuoka, Japan. Med Sci Sports Exerc. 1999 Feb;31(2):342–7

We have developed a sandwich immunoassay method for quantitatively measuring site-specific phosphorylation of multiple proteins and at multiple sites within a protein simultaneously, without the need for multiple, phospho-site-specific detection antibodies. This approach utilizes antibodies to capture a unique peptide sequence (EpiTag) proximal to the phosphorylation site of interest. The captured peptides containing the phosphorylation site(s) of interest are then interrogated with a commercially available, pan-anti-phospho antibody to determine whether the site is indeed phosphorylated. Unlike traditional immunoassays, select epitopes are made available to the antibodies by a unique sample-processing procedure. The procedure involves proteolytic fragmentation of proteins in order to segregate multiple phosphorylation sites within a given protein. Since proteolytic cleavage is predictable, quantitation is achieved by interpolating protein concentrations from standard curves generated with synthetic phosphopeptide standards. This approach obviates the need for phosphoproteins, which can be difficult to generate and qualify. The sandwich immunoassays demonstrate titration over a large dynamic range (≥3 logs) and achieve sensitivity at or below picomolar concentrations of standard. In contrast, no sandwich formation is observed with standards that have been dephosphorylated with lambda phosphatase. While the approach is limited to measuring previously discovered phosphorylation sites and does not distinguish between multiple sites that lie within the same proteolytic fragment, this proprietary approach is directly applicable to both bead-based and planar arrays, and has been multiplexed and used to measure >30 phosphotyrosine sites on various targets across multiple signaling pathways. In addition, the approach is currently being evaluated for multiplexed measurements of other phosphoresidues, and we believe this technology can be applied specifically, to study and quantify particular signaling events, as well as on a more global level, to identify and “fingerprint” activated signaling pathways during different disease states or in response to therapeutic strategies.

Although obesity affects all cultures, ethnic groups and social strata, this disorder affects African Americans, Hispanics and the poor at a disproportionate rate. The Downstart Pediatric Healthy Lifestyle Program was developed to provide a multi-disciplinary behavioral modification program for inner city families in Brooklyn, New York interested in leading a healthier, more active lifestyle. The Downstart Program uses a four-pronged approach of medical evaluation, exercise, nutritional education and lifestyle modification. A psychological evaluation is performed to determine the individual's ability and readiness to participate in group activities. Baseline physical fitness, flexibility and muscle strength are measured, followed by a twice-weekly karate/martial arts/dance program, incorporating principles established by the President's Council on Exercise. Nutritional and behavioral modification aspects of the program consist of weekly education about food groups, portion control, goal setting and appropriate rewards for attaining goals. Our preliminary results indicate that the Downstart Program may be a viable intervention for weight loss. Further study is needed to improve strategies for motivating patients and means and criteria for assessing long-term effects on health and lifestyle.