Sonic Hedgehog activates prostaglandin signaling to stabilize primary cilium length.

Sonic Hedgehog (SHH) is a driver of embryonic patterning that, when corrupted, triggers developmental disorders and cancers. SHH effector responses are organized through primary cilia (PC) that grow and retract with the cell cycle and in response to extracellular cues. Disruption of PC homeostasis corrupts SHH regulation, placing significant pressure on the pathway to maintain ciliary fitness. Mechanisms by which ciliary robustness is ensured in SHH-stimulated cells are not yet known. Herein, we reveal a crosstalk circuit induced by SHH activation of Phospholipase A2α that drives ciliary E-type prostanoid receptor 4 (EP4) signaling to ensure PC function and stabilize ciliary length. We demonstrate that blockade of SHH-EP4 crosstalk destabilizes PC cyclic AMP (cAMP) equilibrium, slows ciliary transport, reduces ciliary length, and attenuates SHH pathway induction. Accordingly, Ep4-/- mice display shortened neuroepithelial PC and altered SHH-dependent neuronal cell fate specification. Thus, SHH initiates coordination between distinct ciliary receptors to maintain PC function and length homeostasis for robust downstream signaling.

Stability and Reactivity of Aromatic Radical Anions in Solution with Relevance to Birch Reduction.

We investigate the electronic structure of aromatic radical anions in the solution phase employing a combination of liquid-jet (LJ) photoelectron (PE) spectroscopy measurements and electronic structure calculations. By using recently developed protocols, we accurately determine the vertical ionization energies of valence electrons of both the solvent and the solute molecules. In particular, we first characterize the pure solvent of tetrahydrofuran (THF) by LJ-PE measurements in conjunction with ab initio molecular dynamics simulations and G0W0 calculations. Next, we determine the electronic structure of neutral naphthalene (Np) and benzophenone (Bp) as well as their radical anion counterparts Np- and Bp- in THF. Wherever feasible, we performed orbital assignments of the measured PE features of the aromatic radical anions, with comparisons to UV-vis absorption spectra of the corresponding neutral molecules being instrumental in rationalizing the assignments. Analysis of the electronic structure differences between the neutral species and their anionic counterparts provides understanding of the primarily electrostatic stabilization of the radical anions in solution. Finally, we obtain a very good agreement of the reduction potentials extracted from the present LJ-PES measurements of Np- and Bp- in THF with previous electrochemical data from cyclic voltammetry measurements. In this context, we discuss how the choice of solvent holds significant implications for optimizing conditions for the Birch reduction process, wherein aromatic radical anions play crucial roles as reactive intermediates.

Ascorbic acid modulates the structure of the Pseudomonas aeruginosa virulence factor pyocyanin and ascorbic acid-furanone-30 combination facilitate biofilm disruption.

The production of pyocyanin by Pseudomonas aeruginosa increases its virulence, fitness and biofilm formation. Pyocyanin is also a redox molecule and we hypothesize that ascorbic acid being an antioxidant will interact with pyocyanin. The main objective of this study was to investigate the potential interaction of ascorbic acid with pyocyanin, and also to investigate the impact of ascorbic acid in combination with Furanone-30 on quorum sensing and biofilm formation of P. aeruginosa. When incubated with ascorbic acid, hyperchromic and hypsochromic shifts in pyocyanin absorbance peaks at 385 nm and 695 nm were observed. In the presence of dehydroascorbic acid and citric acid, these shifts were absent, indicating that the intrinsic antioxidant property of ascorbic acid was probably essential in binding to pyocyanin. NMR spectroscopy showed shifts in 1H NMR pyocyanin peaks between 8.2 to 5.8 ppm when incubated in the presence of ascorbic acid. Density Functional Theory (DFT) supported potential interactions between the -CH2OH or -OH moieties of ascorbic acid with the -C=O moiety of pyocyanin. The pyocyanin-ascorbic acid complex impaired pyocyanin binding to DNA. Ascorbic acid combined with furanone-30 elevated quorum-sensing inhibition in P. aeruginosa, which was directly associated with significantly reduced P. aeruginosa virulence, adhesion, aggregation and biofilm formation and enhanced antibiotic-mediated bacterial killing. This study demonstrated that the antioxidant ascorbic acid directly binds to pyocyanin, modulates its structure and results in disruption of biofilm formation and associated tolerance to antibiotics.

Targeting KDM4 for treating PAX3-FOXO1-driven alveolar rhabdomyosarcoma.

Chimeric transcription factors drive lineage-specific oncogenesis but are notoriously difficult to target. Alveolar rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue sarcoma transformed by the pathognomonic Paired Box 3-Forkhead Box O1 (PAX3-FOXO1) fusion protein, which governs a core regulatory circuitry transcription factor network. Here, we show that the histone lysine demethylase 4B (KDM4B) is a therapeutic vulnerability for PAX3-FOXO1+ RMS. Genetic and pharmacologic inhibition of KDM4B substantially delayed tumor growth. Suppression of KDM4 proteins inhibited the expression of core oncogenic transcription factors and caused epigenetic alterations of PAX3-FOXO1-governed superenhancers. Combining KDM4 inhibition with cytotoxic chemotherapy led to tumor regression in preclinical PAX3-FOXO1+ RMS subcutaneous xenograft models. In summary, we identified a targetable mechanism required for maintenance of the PAX3-FOXO1-related transcription factor network, which may translate to a therapeutic approach for fusion-positive RMS.

Recent Advances with KDM4 Inhibitors and Potential Applications.

The histone lysine demethylase 4 (KDM4) family plays an important role in regulating gene transcription, DNA repair, and metabolism. The dysregulation of KDM4 functions is associated with many human disorders, including cancer, obesity, and cardiovascular diseases. Selective and potent KDM4 inhibitors may help not only to understand the role of KDM4 in these disorders but also to provide potential therapeutic opportunities. Here, we provide an overview of the field and discuss current status, challenges, and opportunities lying ahead in the development of KDM4-based anticancer therapeutics.

A WIN Consortium phase I study exploring avelumab, palbociclib, and axitinib in advanced non-small cell lung cancer.

BACKGROUND: The Worldwide Innovative Network (WIN) Consortium has developed the Simplified Interventional Mapping System (SIMS) to better define the cancer molecular milieu based on genomics/transcriptomics from tumor and analogous normal tissue biopsies. SPRING is the first trial to assess a SIMS-based tri-therapy regimen in advanced non-small cell lung cancer (NSCLC). METHODS: Patients with advanced NSCLC (no EGFR, ALK, or ROS1 alterations; PD-L1 unrestricted; ≤2 prior therapy lines) received avelumab, axitinib, and palbociclib (3 + 3 dose escalation design). RESULTS: Fifteen patients were treated (five centers, four countries): six at each of dose levels 1 (DL1) and DL2; three at DL3. The most common ≥Grade 3 adverse events were neutropenia, hypertension, and fatigue. The recommended Phase II dose (RP2D) was DL1: avelumab 10 mg/kg IV q2weeks, axitinib 3 mg po bid, and palbociclib 75 mg po daily (7 days off/21 days on). Four patients (27%) achieved a partial response (PR) (progression-free survival [PFS]: 14, 24, 25 and 144+ weeks), including two after progression on pembrolizumab. Four patients attained stable disease (SD) that lasted ≥24 weeks: 24, 27, 29, and 64 weeks. At DL1 (RP2D), four of six patients (66%) achieved stable disease (SD) ≥6 months/PR (2 each). Responders included patients with no detectable PD-L1 expression and low tumor mutational burden. CONCLUSIONS: Overall, eight of 15 patients (53%) achieved clinical benefit (SD ≥ 24 weeks/PR) on the avelumab, axitinib, and palbociclib combination. This triplet showed antitumor activity in NSCLC, including in tumors post-pembrolizumab progression, and was active at the RP2D, which was well tolerated. NCT03386929 clinicaltrial.gov.

Phenyl-Glutarimides: Alternative Cereblon Binders for the Design of PROTACs.

Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at low picomolar concentrations (IC50 =3 pM; BRD4 DC50 =0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.

Intra- and Interlaboratory Reproducibility of the Sensitivity to Endocrine Therapy Assay for Stage II/III Breast Cancer.

BACKGROUND: The sensitivity to endocrine therapy assay (SET2,3) predicts treatment outcomes in Stage II-III breast cancer. SET2,3 measures transcription related to estrogen and progesterone receptors (SETER/PR index) and the molecular subtype (RNA4: ESR1, PGR, ERBB2, AURKA) from formalin-fixed paraffin-embedded (FFPE) tissue sections. METHODS: We designed a nested study across 3 pathology laboratories, each testing 60 breast cancers twice in controlled batches. Laboratories macrodissected and directly homogenized the unstained FFPE tumor sections, then performed the QuantiGene Plex bead-based hybridization assay. SET2,3 was calculated centrally using predefined statistical R-scripts and applying pre-defined cutpoints. Concordance correlation coefficient (CCC) was calculated from continuous measurements and Kappa statistic from categorical results. A mixed-effects model estimated contributions to bias (fixed effects) and variance (random effects) from the replicated design. RESULTS: Intralaboratory (CCC 0.96-0.99) and interlaboratory (CCC 0.98-0.99) SET2,3 results were concordant, with rates of agreement for high/low categorization within (Kappa 0.83-0.93) and between laboratories (Kappa 0.87-0.88). The relative contributions to overall variance of SET2,3 measurements were 96.90% from biological differences between cancers, 0.67% from interlaboratory variability, and 2.44% from residual causes including intralaboratory replicates. Similar results were obtained with SETER/PR, the baseline prognostic index calculated using pathological or clinical tumor and nodal staging information, and the 4 individual genes (ESR1, PGR, ERBB2, and AURKA). CONCLUSION: Intra- and interpathology laboratory measurements of SET2,3 and its components were highly reproducible when tested from FFPE tumor sections.

Synthesis of Isotopically Labeled, Spin-Isolated Tyrosine and Phenylalanine for Protein NMR Applications.

Isotopically labeled amino acids are widely used to study the structure and dynamics of proteins by NMR. Herein we describe a facile, gram-scale synthesis of compounds 1b and 2b under standard laboratory conditions from the common intermediate 7. 2b is obtained via simple deprotection, while 1b is accessed through a reductive deoxygenation/deuteration sequence and deprotection. 1b and 2b provide improved signal intensity using lower amounts of labeled precursor and are alternatives to existing labeling approaches.

Identification of Potent, Selective, and Orally Bioavailable Small-Molecule GSPT1/2 Degraders from a Focused Library of Cereblon Modulators.

Whereas the PROTAC approach to target protein degradation greatly benefits from rational design, the discovery of small-molecule degraders relies mostly on phenotypic screening and retrospective target identification efforts. Here, we describe the design, synthesis, and screening of a large diverse library of thalidomide analogues against a panel of patient-derived leukemia and medulloblastoma cell lines. These efforts led to the discovery of potent and novel GSPT1/2 degraders displaying selectivity over classical IMiD neosubstrates, such as IKZF1/3, and high oral bioavailability in mice. Taken together, this study offers compound 6 (SJ6986) as a valuable chemical probe for studying the role of GSPT1/2 in vitro and in vivo, and it supports the utility of a diverse library of CRBN binders in the pursuit of targeting undruggable oncoproteins.

17-DMAG dually inhibits Hsp90 and histone lysine demethylases in alveolar rhabdomyosarcoma.

Histone lysine demethylases (KDMs) play critical roles in oncogenesis and therefore may be effective targets for anticancer therapy. Using a time-resolved fluorescence resonance energy transfer demethylation screen assay, in combination with multiple orthogonal validation approaches, we identified geldanamycin and its analog 17-DMAG as KDM inhibitors. In addition, we found that these Hsp90 inhibitors increase degradation of the alveolar rhabdomyosarcoma (aRMS) driver oncoprotein PAX3-FOXO1 and induce the repressive epigenetic mark H3K9me3 and H3K36me3 at genomic loci of PAX3-FOXO1 targets. We found that as monotherapy 17-DMAG significantly inhibits expression of PAX3-FOXO1 target genes and multiple oncogenic pathways, induces a muscle differentiation signature, delays tumor growth and extends survival in aRMS xenograft mouse models. The combination of 17-DMAG with conventional chemotherapy significantly enhances therapeutic efficacy, indicating that targeting KDM in combination with chemotherapy may serve as a therapeutic approach to PAX3-FOXO1-positive aRMS.

Structure and mechanism of Staphylococcus aureus oleate hydratase (OhyA).

Flavin adenine dinucleotide (FAD)-dependent bacterial oleate hydratases (OhyAs) catalyze the addition of water to isolated fatty acid carbon-carbon double bonds. Staphylococcus aureus uses OhyA to counteract the host innate immune response by inactivating antimicrobial unsaturated fatty acids. Mechanistic information explaining how OhyAs catalyze regiospecific and stereospecific hydration is required to understand their biological functions and the potential for engineering new products. In this study, we deduced the catalytic mechanism of OhyA from multiple structures of S. aureus OhyA in binary and ternary complexes with combinations of ligands along with biochemical analyses of relevant mutants. The substrate-free state shows Arg81 is the gatekeeper that controls fatty acid entrance to the active site. FAD binding engages the catalytic loop to simultaneously rotate Glu82 into its active conformation and Arg81 out of the hydrophobic substrate tunnel, allowing the fatty acid to rotate into the active site. FAD binding also dehydrates the active site, leaving a single water molecule connected to Glu82. This active site water is a hydronium ion based on the analysis of its hydrogen bond network in the OhyA•PEG400•FAD complex. We conclude that OhyA accelerates acid-catalyzed alkene hydration by positioning the fatty acid double bond to attack the active site hydronium ion, followed by the addition of water to the transient carbocation intermediate. Structural transitions within S. aureus OhyA channel oleate to the active site, curl oleate around the substrate water, and stabilize the hydroxylated product to inactivate antimicrobial fatty acids.

Bromodomain-Selective BET Inhibitors Are Potent Antitumor Agents against MYC-Driven Pediatric Cancer.

Inhibition of members of the bromodomain and extraterminal (BET) family of proteins has proven a valid strategy for cancer chemotherapy. All BET identified to date contain two bromodomains (BD; BD1 and BD2) that are necessary for recognition of acetylated lysine residues in the N-terminal regions of histones. Chemical matter that targets BET (BETi) also interact via these domains. Molecular and cellular data indicate that BD1 and BD2 have different biological roles depending upon their cellular context, with BD2 particularly associated with cancer. We have therefore pursued the development of BD2-selective molecules both as chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of tetrahydroquinoline analogs that exhibit >50-fold selectivity for BD2 versus BD1. This selective targeting resulted in engagement with BD-containing proteins in cells, resulting in modulation of MYC proteins and downstream targets. These compounds were potent cytotoxins toward numerous pediatric cancer cell lines and were minimally toxic to nontumorigenic cells. In addition, unlike the pan BETi (+)-JQ1, these BD2-selective inhibitors demonstrated no rebound expression effects. Finally, we report a pharmacokinetic-optimized, metabolically stable derivative that induced growth delay in a neuroblastoma xenograft model with minimal toxicity. We conclude that BD2-selective agents are valid candidates for antitumor drug design for pediatric malignancies driven by the MYC oncogene. SIGNIFICANCE: This study presents bromodomain-selective BET inhibitors that act as antitumor agents and demonstrates that these molecules have in vivo activity towards neuroblastoma, with essentially no toxicity.

A Small Hypoxia Signature Predicted pCR Response to Bevacizumab in the Neoadjuvant GeparQuinto Breast Cancer Trial.

PURPOSE: In breast cancer, bevacizumab increased pCR rate but not long-term survival and no predictive markers are available to identify patients with long-term benefit from the drug. EXPERIMENTAL DESIGN: We profiled 289 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) biopsies of HER2-negative patients from the GeparQuinto trial of neoadjuvant chemotherapy ± bevacizumab by exome-capture RNA-sequencing (RNA-seq). In a prospectively planned study, we tested molecular signatures for response prediction. IHC validation was performed using tissue microarrays. RESULTS: We found strong agreement of molecular and pathologic parameters as hormone receptors, grading, and lymphocyte infiltration in 221 high-quality samples. Response rates (49.3% pCR overall) were higher in basal-like (68.9%) and HER2-enriched (45.5%) than in luminal B (35.7%), luminal A (17.9%), and normal-like (20.0%) subtypes. T-cell (OR = 1.60; 95% confidence interval, 1.21-2.12; P = 0.001), proliferation (OR = 2.88; 95% CI, 2.00-4.15; P < 0.001), and hypoxia signatures (OR = 1.92; 95% CI, 1.41-2.60; P < 0.001) significantly predicted pCR in univariate analysis. In a prespecified multivariate logistic regression, a small hypoxia signature predicted pCR (OR = 2.40; 95% CI, 1.28-4.51; P = 0.006) with a significant interaction with bevacizumab treatment (P = 0.020). IHC validation using NDRG1 as marker revealed highly heterogenous expression within tissue leading to profound loss of sensitivity in TMA analysis, still a significant predictive value for pCR was detected (P = 0.025). CONCLUSIONS: Exome-capture RNA-seq characterizes small FFPE core biopsies by reliably detecting factors as for example ER status, grade, and tumor-infiltrating lymphocytes levels. Beside molecular subtypes and immune signatures, a small hypoxia signature predicted pCR to bevacizumab, which could be validated by IHC. The signature can have important applications for bevacizumab treatment in different cancer types and might also have a role for novel combination therapies of bevacizumab with immune checkpoint inhibition.

Development and optimization of OspC chimeritope vaccinogens for Lyme disease.

Experimental Outer surface protein (Osp) C based subunit chimeritope vaccinogens for Lyme disease (LD) were assessed for immunogenicity, structure, ability to elicit antibody (Ab) responses to divergent OspC proteins, and bactericidal activity. Chimeritopes are chimeric epitope based proteins that consist of linear epitopes derived from multiple proteins or multiple variants of a protein. An inherent advantage to chimeritope vaccinogens is that they can be constructed to trigger broadly protective Ab responses. Three OspC chimeritope proteins were comparatively assessed: Chv1, Chv2 and Chv3. The Chv proteins possess the same set of 18 linear epitopes derived from 9 OspC type proteins but differ in the physical ordering of epitopes or by the presence or absence of linkers. All Chv proteins were immunogenic in mice and rats eliciting high titer Ab. Immunoblot and enzyme linked immunosorbent assays demonstrated that the Chv proteins elicit IgG that recognizes a diverse array of OspC type proteins. The panel included OspC proteins produced by N. American and European strains of the LD spirochetes. Rat anti-Chv antisera uniformly labeled intact, non-permeabilized Borreliella burgdorferi demonstrating that vaccinal Ab can bind to targets that are naturally presented on the spirochete cell surface. Vaccinal Ab also displayed potent complement dependent-Ab mediated killing activity. This study highlights the ability of OspC chimeritopes to serve as vaccinogens that trigger potentially broadly protective Ab responses. In addition to the current use of an OspC chimeritope in a canine LD vaccine, chimeritopes can serve as key components of human LD subunit vaccines.

The Enriched Life Scale (ELS): Development, exploratory factor analysis, and preliminary construct validity for U.S. military veteran and civilian samples.

The U.S. military veteran serving nonprofit, Team Red, White & Blue (RWB), defined an "enriched life" as having physical, mental, and emotional health; supportive relationships; and a sense of purpose. Until now, no corresponding measure of an "enriched life" existed for the purposes of clinical assessment and research. The primary objective of this study was to explore the psychometric properties of the Enriched Life Scale (ELS) in veteran and civilian samples. Our secondary objective was to examine differences in ELS scores in subgroups of veterans who had combat deployments and service-related injuries. Veteran thought leaders working with social scientists developed the ELS and implemented exploratory factor analysis to determine the underlying dimensions of the "enriched life" construct. One thousand one hundred and eighty-seven veterans and 598 civilians participated in the study. This article describes the development of the ELS, reliability, exploratory factor analysis, and preliminary construct validity. The final ELS had 40 items and consisted of five constructs that were labeled "Genuine Relationships"; "Sense of Purpose"; "Engaged Citizenship"; "Mental Health"; and "Physical Health." Measures had high internal consistency (α = 0.82-0.94). Civilians scored higher than veterans on every ELS item, subscales, and total score, with small to large effect size differences noted between groups. In the veteran subsample, individuals with combat experience scored lower on every ELS subscale than those without combat experience (small effect sizes), as did veterans with service-related injuries in comparison to those without them (small to large effect sizes). This article establishes preliminary psychometric properties of the ELS-a promising instrument to measure an enriched life. Further study is currently underway to establish confirmatory factor analyses and explore extending its usage to diverse military and civilian samples.

Confirmatory Factor Analysis of the Enriched Life Scale Among US Military Veterans.

The Enriched Life Scale (ELS) is a 40-item measure developed by the military veteran service organization, Team Red, White & Blue (RWB), to systematically capture and quantify the lived experiences of military veterans transitioning to civilian life. As Team RWB's mission is to "enrich veterans' lives," veterans who conceived of and co-developed the ELS as a psychometric instrument defined what an "enriched life" would entail. Exploratory factor analysis (EFA) of the ELS revealed a five-factor structure capturing the domains of: physical health, mental health, genuine relationships, sense of purpose, and engaged citizenship. The goal of the current study was to use confirmatory factor analysis to validate the factor structure of the ELS in a sample of veterans not affiliated with Team RWB. We also sought to explore convergent validity with the Military to Civilian Questionnaire, a measure of military to civilian reintegration challenges. Five hundred and twenty-nine veterans participated in the study. We estimated three models, one-factor, four-factor, and five-factor model via maximum likelihood estimation with robust Huber-White standard errors. The five-factor model showed the best fit to the data (RMSEA = 0.05, CFI = 0.90, TLI = 0.90, SRMR = 0.06). Additionally, the five-factor model demonstrated convergent and discriminant validity, as well as internal consistency reliability (genuine relationships, α = 0.90; sense of purpose, α = 0.93; engaged citizenship, α = 0.89; mental health, α = 0.88; and physical health, α = 0.78). Overall, the ELS is a valid and reliable measure of veteran enrichment and could potentially be used in conjunction with diagnostic instruments that capture strain-related transition challenges (to include mental health disorders) to capture post-military service wellbeing.

1H, 13C, and 15N resonance assignments of N-acetylmuramyl-L-alanine amidase (AmiC) N-terminal domain (NTD) from Neisseria gonorrhoeae.

Gonorrhea infections are becoming more difficult to treat due to the prevalence of strains exhibiting resistance to antibiotics and new therapeutic approaches are needed. N-acetylmuramyl-L-alanine amidase (AmiC) from Neisseria gonorrhoeae is a hydrolase that functions during cell division by cleaving the bond between the N-acetylmuramyl and L-alanine moieties of peptidoglycan. Inhibiting this enzyme offers the prospect of restoring the efficacy of existing antibiotics as treatments against N. gonorrhoeae. Of its two domains, the C-terminal domain catalyses the hydrolysis reaction and the N-terminal domain (NTD) is believed to target AmiC to its peptidoglycan substrate. Here, we report the 1H, 13C, and 15N resonance assignments of a 131 amino acid NTD construct of AmiC by heteronuclear NMR spectroscopy. The assignments represent the first for N. gonorrhoeae AmiC-NTD, laying the groundwork for detailed examination of its structure and dynamics, and providing a platform for new drug discovery efforts to address antimicrobial-resistant N. gonorrhoeae.

Regional differences in BMI, obesity, and exercise frequency in a large veteran service organization: A secondary analysis of new veteran member surveys from Team Red, White & Blue.

The purpose of the present study was to examine regional differences in average self-reported BMI, obesity prevalence, and frequent exercise (FE) among members of Team Red, White, and Blue (Team RWB) - a military veteran service organization founded to increase physical activity in veterans. A total of 10,015 military veterans participated in a needs assessment conducted by Team RWB between December 2014 and August 2016. Multivariate regression analysis with bootstrapped coefficients revealed that: BMI was highest in the Midwest region (M = 28.282) of the United States, F(20, 9882) = 105.560, p < 0.001; obesity prevalence was highest in the Southcentral (32.300%) and Southeast (32.200%) regions, x2(9731) = 10,850, p < 0.001; and FE was most prevalent in the Mid-Atlantic region (67.3%), x2(9882) = 11,291, p < 0.001.The results of this study closely mirror results found in studies of the general population. A better understanding of the geographic distribution of these outcomes could guide the targeting of sub-populations for public health programs. In particular, Team Red, White & Blue community growth and other fitness based public health programs could be expanded to reach more veterans.