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Recent data show that extracellular signals are transmitted through a network of proteins rather than hierarchical signaling pathways, suggesting that the inhibition of a single component of a canonical pathway is insufficient for the treatment of cancer. The biologic outcome of signaling through a network is inherently more robust and resistant to inhibition of a single network component. In this study, we conducted a functional chemical genetic screen to identify novel interactions between signaling inhibitors that would not be predicted on the basis of our current understanding of signaling networks. We screened over 300 drug combinations in nine melanoma cell lines and have identified pairs of compounds that show synergistic cytotoxicity. The synergistic cytotoxicities identified did not correlate with the known RAS and BRAF mutational status of the melanoma cell lines. Among the most robust results was synergy between sorafenib, a multikinase inhibitor with activity against RAF, and diclofenac, a nonsteroidal anti-inflammatory drug (NSAID). Drug substitution experiments using the NSAIDs celecoxib and ibuprofen or the MAP–ERK kinase inhibitor PD325901 and the RAF inhibitor RAF265 suggest that inhibition of COX and mitogen-activated protein kinase signaling are targets for the synergistic cytotoxicity of sorafenib and diclofenac. Cotreatment with sorafenib and diclofenac interrupts a positive feedback signaling loop involving extracellular signal–regulated kinase, cellular phospholipase A2, and COX. Genome-wide expression profiling shows synergy-specific downregulation of survival-related genes. This study has uncovered novel functional drug combinations and suggests that the underlying signaling networks that control responses to targeted agents can vary substantially, depending on unexplored components of the cell genotype. Mol Cancer Ther; 11(11); 2505–15. ©2012 AACR.

Supplementary Table 1 from Compensatory Pathways Induced by MEK Inhibition Are Effective Drug Targets for Combination Therapy against Castration-Resistant Prostate Cancer

Supplementary Figure and Table Legend from Compensatory Pathways Induced by MEK Inhibition Are Effective Drug Targets for Combination Therapy against Castration-Resistant Prostate Cancer

PDF file, 428K, The combination of sorafenib and diclofenac causes cell death.

Targeted therapies have often given disappointing results when used as single agents in solid tumors, suggesting the importance of devising rational combinations of targeted drugs. We hypothesized that construction of such combinations could be guided by identification of growth and survival pathways whose activity or expression become upregulated in response to single-agent drug treatment. We mapped alterations in signaling pathways assessed by gene array and protein phosphorylation to identify compensatory signal transduction pathways in prostate cancer xenografts treated with a MAP/ERK kinase (MEK) inhibitor PD325901. In addition to numerous components of the extracellular signal–regulated kinase (ERK) signaling pathway, components of the IKK, hedgehog, and phosphoinositide 3-kinase/Akt/mTOR pathways were upregulated following treatment with PD325901. Combinations of PD325901 with inhibitors of any one of these upregulated pathways provided synergistically greater growth inhibition of in vitro cell growth and survival than the individual drugs alone. Thus, the identification of compensatory signal transduction pathways paves the way for rational combinatorial therapies for the effective treatment of prostate cancer. Mol Cancer Ther; 10(9); 1581–90. ©2011 AACR.

Supplemental text for Methods and Results.

Blood cell counts for all patients on study, except lymphocytes.

PDF file - 155K

Detail of treatment-related toxicities.

PDF file - 126K, Detailed data are shown for selected proteins assayed by RPPA.

PDF file - 39K, Lymphocyte counts for all patients on study - detail.

Purpose: A CTEP-sponsored phase II trial was conducted to evaluate safety and clinical activity of combination therapy with CCI-779 (temsirolimus) and bevacizumab in patients with advanced melanoma.Experimental Design: Patients with unresectable stage III to IV melanoma were treated intravenously with temsirolimus 25 mg weekly and bevacizumab 10 mg every 2 weeks. Adverse events were recorded using CTCAE v3.0. Tumor response was assessed by Response Evaluation Criteria in Solid Tumors and overall survival was recorded. Correlative studies measured protein kinases and histology of tumor biopsies and immune function in peripheral blood.Results: Seventeen patients were treated. Most patients tolerated treatment well, but 2 had grade 4 lymphopenia and 1 developed reversible grade 2 leukoencephalopathy. Best clinical response was partial response (PR) in 3 patients [17.7%, 90% confidence interval (CI) 5, 0–39.6], stable disease at 8 weeks (SD) in 9 patients, progressive disease (PD) in 4 patients, and not evaluable in 1 patient. Maximal response duration for PR was 35 months. Ten evaluable patients had BRAFWT tumors, among whom 3 had PRs, 5 had SD, and 2 had PD. Correlative studies of tumor biopsies revealed decreased phospho-S6K (d2 and d23 vs. d1, P < 0.001), and decreased mitotic rate (Ki67+) among melanoma cells by d23 (P = 0.007). Effects on immune functions were mixed, with decreased alloreactive T-cell responses and decreased circulating CD4+FoxP3+ cells.Conclusion: These data provide preliminary evidence for clinical activity of combination therapy with temsirolimus and bevacizumab, which may be greater in patients with BRAFwt melanoma. Mixed effects on immunologic function also support combination with immune therapies. Clin Cancer Res; 19(13); 3611–20. ©2013 AACR.

PDF file - 208K, Blood cell counts for all patients on study, except lymphocytes.

Detailed data are shown for selected proteins assayed by RPPA.

Lymphocyte counts for all patients on study - detail

We collected fishes and habitat data at 138 streams to evaluate the effects of introduced brown trout ( Salmo trutta) and habitat conditions on occurrence, detection, abundance, and size structure of sculpin ( Cottus spp.), longnose dace ( Rhinichthys cataractae), and southern redbelly dace ( Chrosomus erythrogaster) in the Driftless Area, USA. Sculpin detection decreased with increasing stream velocity, whereas southern redbelly dace detection increased with stream depth. Sculpin occupancy declined with increasing stream temperature and velocity and increased with increasing forested land, boulder substrate, and brown trout length and abundance. Longnose and southern redbelly dace occupancy and abundance declined with increasing brown trout abundance and occupancy increased with stream temperature. Longnose dace occupancy also increased with increasing stream temperature and cobble substrate and declined with increasing elevation. Native fish size structure was unrelated to brown trout presence. Our results suggest that effects of brown trout are not ubiquitous across native fishes and depend on abiotic conditions and species-specific habitat requirements, highlighting the need to consider both biotic and abiotic conditions when balancing native species conservation with introduced sportfish management.

Popularity of bass (Micropterus spp.) catch and release and tournament angling during the past decade has resulted in increased potential for these activities to induce population level effects. Understanding capture rates and mortality sources relative to total population mortality is essential to focus of management. We conducted monthly electrofishing, solicited non-tournament angler tag returns, and censused largemouth bass (Micropterus salmoides) tournaments at Brushy Creek Lake, Iowa, USA, from April 2015 to June 2018. We used a multistate mark–recapture model to evaluate the effects of air temperature, water temperature, tournament bass per angler, and tournament initial mortality on non-tournament and tournament angler capture probability and natural, non-tournament angling, and initial and delayed tournament mortality. Average total annual mortality was 0.66 with natural mortality representing the largest mortality source (0.57) followed by delayed tournament mortality (0.06), non-tournament angling mortality (0.02), and initial tournament mortality (0.006). Our results reveal both non-tournament and tournament angling mortality are low compared to natural mortality in some lakes. Therefore, cumulative angling mortality likely has minimal population level effects on some bass populations.

Largemouth Bass Micropterus nigricans anglers have mainly adopted catch-and-release practices with limited direct fishing mortality. However, catch-and-release angling could cause delayed mortality and non-lethal effects that could potentially impact population dynamics. For instance, hooking can result in jaw deformities, with unknown subsequent effects on vulnerability to angling, condition, growth, and mortality. Our objectives were to assess the prevalence of Largemouth Bass jaw deformities and test for differences in angling vulnerability, growth, condition, size structure, and mortality of fish with and without jaw deformities. Largemouth Bass were captured by electrofishing and by anglers at 41 tournaments at Brushy Creek, Iowa between April and August 2015. Jaw deformities were observed in 7.0% of fish caught at tournaments and 3.3% caught while electrofishing (5.8% overall). Angling recapture rates of tagged fish with (78.9%) and without (76.8%) jaw deformities were similar. Condition, growth, size structure, and mortality of fish with and without jaw deformities were also similar. Our results indicate jaw deformities have little effect on Largemouth Bass angling vulnerability, condition, growth, or survival; however, we still recommend careful handling of fish to minimize injuries that may occur during angling.

The Bcl-2 inhibitor venetoclax has yielded exceptional clinical responses in chronic lymphocytic leukemia (CLL). However, de novo resistance can result in failure to achieve negative minimal residual disease and predicts poor treatment outcomes. Consequently, additional proapoptotic drugs, such as inhibitors of Mcl-1 and Bcl-xL, are in development. By profiling antiapoptotic proteins using flow cytometry, we find that leukemic B cells that recently emigrated from the lymph node (CD69+/CXCR4Low) in vivo are enriched for cell clusters simultaneously overexpressing multiple antiapoptotic proteins (Mcl-1High/Bcl-xLHigh/Bcl-2High) in both treated and treatment-naive CLL patients. These cells exhibited antiapoptotic resistance to multiple BH-domain antagonists, including inhibitors of Bcl-2, Mcl-1, and Bcl-xL, when tested as single agents in a flow cytometry–based functional assay. Antiapoptotic multidrug resistance declines ex vivo, consistent with resistance being generated in vivo by extrinsic microenvironmental interactions. Surviving “persister” cells in patients undergoing venetoclax treatment are enriched for CLL cells displaying the functional and molecular properties of microenvironmentally induced multidrug resistance. Overcoming this resistance required simultaneous inhibition of multiple antiapoptotic proteins, with potential for unwanted toxicities. Using a drug screen performed using patient peripheral blood mononuclear cells cultured in an ex vivo microenvironment model, we identify novel venetoclax drug combinations that induce selective cytotoxicity in multidrug-resistant CLL cells. Thus, we demonstrate that antiapoptotic multidrug-resistant CLL cells exist in patients de novo and show that these cells persist during proapoptotic treatment, such as venetoclax. We validate clinically actionable approaches to selectively deplete this reservoir in patients.

Mercury contamination in aquatic ecosystems is a global concern due to the health risks of consuming contaminated fishes. Fish mercury concentrations are influenced by a range of biotic and abiotic factors that vary among regions, but these complex interactions are difficult to disentangle. We collected bluegill (Lepomis macrochirus), white and black crappie (Pomoxis annularis; P. nigromaculatus), largemouth bass (Micropterus salmoides), walleye (Sander vitreus), muskellunge (Esox masquinongy), and northern pike (E. lucius) from waterbodies throughout Iowa and analyzed them for mercury concentrations. We used land use, water chemistry, and fish characteristics to explain variation in mercury concentrations among and within systems. Mercury concentrations were generally low and undetectable ( 0.05 mg/kg) in 43% of fish analyzed. Detected mercury concentrations were highest in largemouth bass, muskellunge, northern pike, and walleye, lowest in black and white crappie and bluegill, and positively related to fish length and age. Mean lake depth, pH, watershed area to lake area ratio, and percent of watershed as open water were positively related to fish mercury concentrations whereas lake area and percent of watershed as agriculture, developed, forested, and grassland were negatively related to mercury concentrations. Additionally, mercury concentrations were higher in shallow natural lakes compared to other lake types. Our results indicate fish mercury concentrations are lower in Iowa lakes compared to other regions. Models we developed in this study can be used to identify other waterbodies that may have elevated mercury concentrations that can guide fish mercury monitoring programs.

Bighead Carp Hypothalmichthys nobilis and Silver Carp Hypothalmichthys moltrix (hereafter collectively referred to as Bigheaded Carp) have spread throughout the majority of the Mississippi River since the 1970s. The current northern invasion edge of Bigheaded Carp in the Upper Mississippi River (UMR) spans between Pools 14 and 20 because of limited passage at Lock and Dam (LD) 19. Mechanisms limiting adult Bigheaded Carp abundance above LD19 are unknown but may be due in part to lack of reproductive success influenced by adult abundance and environmental factors. Our objective was to investigate how relative adult biomass and river temperature and discharge affect maximum annual Bigheaded Carp larval production in the UMR using a Ricker stock-recruitment model. Adult Bigheaded Carp relative biomass (kg/h) was estimated annually with boat electrofishing and larvae were collected every 10 d between May and August 2014–2017 in Pools 14–20 in the UMR. Adult relative biomass ranged from 0.0 to 880.9 kg/h, whereas maximum annual larval densities ranged from 0.0 to 2,869.4 larvae/m3. After accounting for variability among pools and years, the most supported linear Ricker stock-recruitment model indicated the number of recruits per spawner decreased with increasing adult relative biomass and increased with mean discharge. Our results highlight the importance of adult biomass and river discharge conditions for reproduction of Bigheaded Carp along leading edges of invasion. Management strategies that aim to maintain low adult abundance where reproduction is not yet occurring could help limit population increases via reproduction, whereas reducing high adult biomass (e.g., commercial harvest, barriers) may result in greater Bigheaded Carp reproductive output in the UMR.

Invasive Bighead Carp Hypophthalmichthys nobilis and Silver Carp H. molitrix have infested and caused largescale ecological and economic damage to the Illinois, Mississippi, and Ohio rivers. We compiled demographic data from 42,995 fish from 23 pools in the Illinois, Mississippi, and Ohio rivers, which universities and management agencies previously collected as part of management, monitoring, and research activities. We used this data set to test whether demographic rates (length–weight relations including body condition, mortality, growth curves, and female maturity curves) varied among subpopulations across a gradient of invasion status. We found that length–weight relations and growth curves varied among subpopulations, whereas maturity curves did not. Our findings demonstrated spatial variability in demographic rates for Bighead and Silver carp across a broad geographic area in relation to invasion status and river conditions. Herein, we provide general subpopulation management options and present different hypotheses to explain the observed spatial variability in demographic rates.

Fish hatcheries are generally not adjacent to stocking locations. Thus, hatchery fish undergo transportation processes for variable durations that can result in changes in water quality, fish physiology (e.g., whole blood glucose and plasma cortisol), and reduced survival. Walleye Sander vitreus are commonly stocked throughout North America with variable stocking success, possibly due to altered physiological responses associated with changes in water quality parameters during transportation. We hypothesized increased transport duration would be associated with increases in water temperature, carbon dioxide, and total ammonia nitrogen and decreases in pH and total alkalinity. We also hypothesized that increases in carbon dioxide, water temperature, un-ionized ammonia, and total ammonia nitrogen would be positively related with Walleye whole blood glucose and plasma cortisol concentrations. Walleye were transported for either 0, 0.5, 3, or 5 h and whole blood glucose and plasma cortisol concentrations and mortality were evaluated for 48 h posttransport. Total ammonia nitrogen concentrations, carbon dioxide, pH, and water temperature increased with transportation duration while total alkalinity decreased. Plasma cortisol and whole blood glucose concentrations of Walleye transported for longer durations took longer to decline relative to those not transported. Water quality parameters were not associated with changes in Walleye whole blood glucose and plasma cortisol concentrations, but they were negatively related with time since transport (P < 0.05). Despite increases in stress, mortality was low (2.5%). Overall, we found evidence to support our hypotheses regarding reduced water quality associated with increasing transport duration. Finally, whole blood glucose and plasma cortisol concentration of Walleye that were transported were similar to those not transported, suggesting handling procedures before transportation could play a significant role in physiological responses measured after transportation. Further, evaluation of stocking procedures aimed at decreasing handling during the loading process could enhance stocking protocols. Elevated concentrations of whole blood glucose and plasma cortisol following transport could make recently stocked Walleye more susceptible to the effects of other environmental factors such as starvation, predation, and disease, all of which could result in increased mortality rates after stocking.