Therapeutics such as lapatinib that target ERBB2 often provide initial clinical benefit but resistance frequently develops. Adaptive responses leading to lapatinib resistance involve reprogramming of ...the kinome through reactivation of ERBB2/ERBB3 signaling and transcriptional upregulation and activation of multiple tyrosine kinases. The heterogeneity of induced kinases prevents their targeting by a single kinase inhibitor, underscoring the challenge of predicting effective kinase inhibitor combination therapies. We hypothesized that to make the tumor response to single kinase inhibitors durable, the adaptive kinome response itself must be inhibited. Genetic and chemical inhibition of BET bromodomain chromatin readers suppresses transcription of many lapatinib-induced kinases involved in resistance including ERBB3, IGF1R, DDR1, MET, and FGFRs, preventing downstream SRC/FAK signaling and AKT reactivation. Combining inhibitors of kinases and chromatin readers prevents kinome adaptation by blocking transcription, generating a durable response to lapatinib and overcoming the dilemma of heterogeneity in the adaptive response.
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Lapatinib induces heterogeneous RTK-based kinome adaptation in ERBB2+ cellsMultiple unrelated kinases contribute to cell growth in the presence of lapatinibBET bromodomain inhibition suppresses expression of lapatinib-induced kinasesTargeting kinome adaptation makes kinase inhibition durable
OBJECTIVES:To describe the quality of life among sepsis survivors.
DESIGN:Secondary analyses of two international, randomized clinical trials (A Controlled Comparison of Eritoran and placebo in ...patients with Severe Sepsis derivation cohort and PROWESS-SHOCK validation cohort).
SETTING:ICUs in North and South America, Europe, Africa, Asia, and Australia.
PATIENTS:Adults with severe sepsis. We analyzed only patients who were functional and living at home without help before sepsis hospitalization (n = 1,143 and 987 from A Controlled Comparison of Eritoran and placebo in patients with Severe Sepsis and PROWESS-SHOCK, respectively).
INTERVENTIONS:None.
MEASUREMENTS AND MAIN RESULTS:In A Controlled Comparison of Eritoran and placebo in patients with Severe Sepsis and PROWESS-SHOCK, the average age of patients living at home independently was 63 and 61 years; 400 (34.9%) and 298 (30.2%) died by 6 months. In A Controlled Comparison of Eritoran and placebo in patients with Severe Sepsis, 580 patients had a quality of life measured using EQ-5D at 6 months. Of these, 41.6% could not live independently (22.7% were home but required help, 5.1% were in nursing home or rehabilitation facilities, and 5.3% were in acute care hospitals). Poor quality of life at 6 months, as evidenced by problems in mobility, usual activities, and self-care domains were reported in 37.4%, 43.7%, and 20.5%, respectively, and the high incidence of poor quality of life was also seen in patients in PROWESS-SHOCK. Over 45% of patients with mobility and self-care problems at 6 months in A Controlled Comparison of Eritoran and placebo in patients with Severe Sepsis died or reported persistent problems at 1 year.
CONCLUSIONS:Among individuals enrolled in a clinical trial who lived independently prior to severe sepsis, one third had died and of those who survived, a further one third had not returned to independent living by 6 months. Both mortality and quality of life should be considered when designing new interventions and considering endpoints for sepsis trials.
IMPORTANCE Eritoran is a synthetic lipid A antagonist that blocks lipopolysaccharide (LPS) from binding at the cell surface MD2-TLR4 receptor. LPS is a major component of the outer membrane of ...gram-negative bacteria and is a potent activator of the acute inflammatory response. OBJECTIVE To determine if eritoran, a TLR4 antagonist, would significantly reduce sepsis-induced mortality. DESIGN, SETTING, AND PARTICIPANTS We performed a randomized, double-blind, placebo-controlled, multinational phase 3 trial in 197 intensive care units. Patients were enrolled from June 2006 to September 2010 and final follow-up was completed in September 2011. INTERVENTIONS Patients with severe sepsis (n = 1961) were randomized and treated within 12 hours of onset of first organ dysfunction in a 2:1 ratio with a 6-day course of either eritoran tetrasodium (105 mg total) or placebo, with n = 1304 and n = 657 patients, respectively. MAIN OUTCOME MEASURES The primary end point was 28-day all-cause mortality. The secondary end points were all-cause mortality at 3, 6, and 12 months after beginning treatment. RESULTS Baseline characteristics of the 2 study groups were similar. In the modified intent-to-treat analysis (randomized patients who received at least 1 dose) there was no significant difference in the primary end point of 28-day all-cause mortality with 28.1% (366/1304) in the eritoran group vs 26.9% (177/657) in the placebo group (P = .59; hazard ratio, 1.05; 95% CI, 0.88-1.26; difference in mortality rate, −1.1; 95% CI, −5.3 to 3.1) or in the key secondary end point of 1-year all-cause mortality with 44.1% (290/657) in the eritoran group vs 43.3% (565/1304) in the placebo group, Kaplan-Meier analysis of time to death by 1 year, P = .79 (hazard ratio, 0.98; 0.85-1.13). No significant differences were observed in any of the prespecified subgroups. Adverse events, including secondary infection rates, did not differ between study groups. CONCLUSIONS AND RELEVANCE Among patients with severe sepsis, the use of eritoran, compared with placebo, did not result in reduced 28-day mortality. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00334828
Protein binding specificities can be manipulated by redesigning contacts that already exist at an interface or by expanding the interface to allow interactions with residues adjacent to the original ...binding site. Previously, we developed a strategy, called AnchorDesign, for expanding interfaces around linear binding epitopes. The epitope is embedded in a loop of a scaffold protein, in our case a monobody, and then surrounding residues on the monobody are optimized for binding using directed evolution or computational design. Using this strategy, we have increased binding affinities by >100-fold, but we have not tested whether it can be used to control protein binding specificities. Here, we test whether AnchorDesign can be used to engineer a monobody that binds specifically to the mitogen-activated protein kinase (MAPK) p38α but not to the related MAPKs ERK2 and JNK. To anchor the binding interaction, we used a small (D) docking motif from the mitogen-activated protein kinase kinase (MAP2K) MKK6 that interacts with similar affinity with p38α and ERK2. Our hypothesis was that by embedding the motif in a larger protein that we could expand the interface and create contacts with residues that are not conserved between p38α and ERK2. Molecular modeling was used to inform insertion of the D motif into the monobody, and a combination of phage and yeast display were used to optimize the interface. Binding experiments demonstrate that the engineered monobody binds to the target surface on p38α and does not exhibit detectable binding to ERK2 or JNK.
The central role of the BRAF-MEK-ERK pathway in controlling cell fate has made this pathway a primary target for deregulated activation in cancer. BRaf is activated by Ras proteins allowing Ras ...oncogenes to constitutively activate the pathway. Activating BRaf mutations are also frequent in several cancers, being the most common oncogenic mutation in thyroid carcinoma and melanoma. There are currently two inhibitors, vemurafenib and dabrafenib, approved for treatment of malignant melanoma having activating BRaf mutations. Concurrent administration of BRAF and MAP-ERK kinase (MEK) inhibitor (trametinib) is significantly more active in patients with BRAF-mutant melanoma than either single agent alone, but progression to resistance ultimately occurs by different mechanisms that increase the activation of extracellular signal-regulated kinase (ERK). Such adaptive changes in tumor cell signaling networks allow bypass of targeted oncoprotein inhibition. This is true with targeted inhibitors for BRaf and MEK as well as specific inhibitors for AKT, mTOR, and many receptor tyrosine kinases such as EGF receptor (EGFR) and HER2. It is this adaptive response to targeted kinase inhibitors that contributes to the failure of single-agent kinase inhibitors to have durable responses. This failure is seen in virtually all cancers treated with single-agent kinase inhibitors, most of which are not as dependent on a single signaling pathway such as BRaf-MEK-ERK in melanoma. Thus, understanding the breadth of adaptive reprogramming responses to specific targeted kinase inhibition will be critical to develop appropriate combination therapies for durable clinical responses.
Neopterin has been extensively used as a clinical marker of immune activation during inflammation in a wide range of conditions and stresses. However, the analysis of neopterin alone neglects the ...cellular reactions that generate it in response to interferon-γ. Neopterin is the oxidation product of 7,8-dihydroneopterin, which is a potent antioxidant generated by interferon-γ-activated macrophages. 7,8-Dihydroneopterin can protect macrophage cells from a range of oxidants through a scavenging reaction that generates either neopterin or dihydroxanthopterin, depending on the oxidant. Therefore, plasma and urinary neopterin levels are dependent on both macrophage activation to generate 7,8-dihydroneopterin and subsequent oxidation to neopterin. This relationship is clearly shown in studies of exercise and impact-induced injury during intense contact sport. Here, we argue that neopterin and total neopterin, which is the combined value of 7,8-dihydroneopterin and neopterin, could provide a more comprehensive analysis of clinical inflammation than neopterin alone.
Although targeted inhibition of oncogenic kinase drivers has achieved remarkable patient responses in many cancers, the development of resistance has remained a significant challenge. Numerous ...mechanisms have been identified, including the acquisition of gatekeeper mutations, activating pathway mutations, and copy number loss or gain of the driver or alternate nodes. These changes have prompted the development of kinase inhibitors with increased selectivity, use of second-line therapeutics to overcome primary resistance, and combination treatment to forestall resistance. In addition to genomic resistance mechanisms, adaptive transcriptional and signaling responses seen in tumors are gaining appreciation as alterations that lead to a phenotypic state change-often observed as an epithelial-to-mesenchymal shift or reversion to a cancer stem cell-like phenotype underpinned by remodeling of the epigenetic landscape. This epigenomic modulation driving cell state change is multifaceted and includes modulation of repressive and activating histone modifications, DNA methylation, enhancer remodeling, and noncoding RNA species. Consequently, the combination of kinase inhibitors with drugs targeting components of the transcriptional machinery and histone-modifying enzymes has shown promise in preclinical and clinical studies. Here, we review mechanisms of resistance to kinase inhibition in cancer, with special emphasis on the rewired kinome and transcriptional signaling networks and the potential vulnerabilities that may be exploited to overcome these adaptive signaling changes.
Targeted therapeutics that are initially effective in cancer patients nearly invariably engender resistance at some stage, an inherent challenge in the use of any molecular-targeted drug in cancer ...settings. In this study, we evaluated resistance mechanisms arising in metastatic melanoma to MAPK pathway kinase inhibitors as a strategy to identify candidate strategies to limit risks of resistance. To investigate longitudinal responses, we developed an intravital serial imaging approach that can directly visualize drug response in an inducible RAF-driven, autochthonous murine model of melanoma incorporating a fluorescent reporter allele (tdTomatoLSL). Using this system, we visualized formation and progression of tumors
, starting from the single-cell level longitudinally over time. Reliable reporting of the status of primary murine tumors treated with the selective MEK1/2 inhibitor (MEKi) trametinib illustrated a time-course of initial drug response and persistence, followed by the development of drug resistance. We found that tumor cells adjacent to bundled collagen had a preferential persistence in response to MEKi. Unbiased transcriptional and kinome reprogramming analyses from selected treatment time points suggested increased c-Kit and PI3K/AKT pathway activation in resistant tumors, along with enhanced expression of epithelial genes and epithelial-mesenchymal transition downregulation signatures with development of MEKi resistance. Similar trends were observed following simultaneous treatment with BRAF and MEK inhibitors aligned to standard-of-care combination therapy, suggesting these reprogramming events were not specific to MEKi alone. Overall, our results illuminate the integration of tumor-stroma dynamics with tissue plasticity in melanoma progression and provide new insights into the basis for drug response, persistence, and resistance.
A longitudinal study tracks the course of MEKi treatment in an autochthonous imageable murine model of melanoma from initial response to therapeutic resistance, offering new insights into the basis for drug response, persistence, and resistance.
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Targeting the dysregulated BRAF-MEK-ERK pathway in cancer has increasingly emerged in clinical trial design. Despite clinical responses in specific cancers using inhibitors targeting BRAF and MEK, ...resistance develops often involving nongenomic adaptive bypass mechanisms. Inhibition of MEK1/2 by trametinib in patients with triple-negative breast cancer (TNBC) induced dramatic transcriptional responses, including upregulation of receptor tyrosine kinases (RTK) comparing tumor samples before and after one week of treatment. In preclinical models, MEK inhibition induced genome-wide enhancer formation involving the seeding of BRD4, MED1, H3K27 acetylation, and p300 that drives transcriptional adaptation. Inhibition of the P-TEFb-associated proteins BRD4 and CBP/p300 arrested enhancer seeding and RTK upregulation. BRD4 bromodomain inhibitors overcame trametinib resistance, producing sustained growth inhibition in cells, xenografts, and syngeneic mouse TNBC models. Pharmacologic targeting of P-TEFb members in conjunction with MEK inhibition by trametinib is an effective strategy to durably inhibit epigenomic remodeling required for adaptive resistance.
Widespread transcriptional adaptation to pharmacologic MEK inhibition was observed in TNBC patient tumors. In preclinical models, MEK inhibition induces dramatic genome-wide modulation of chromatin, in the form of
enhancer formation and enhancer remodeling. Pharmacologic targeting of P-TEFb complex members at enhancers is an effective strategy to durably inhibit such adaptation.
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To evaluate pterins as diagnostic biomarkers of exercise-induced stress.
Systematic review of the literature.
MEDLINE, Scopus and Web of Science were searched in March 2019 for relevant literature. ...We only considered in vivo studies of healthy humans that reported measurement of a pterin(s) in response to exercise or sport with no underlying prior disease or complication. Relevant articles were independently reviewed and resolved by consensus.
We included 29 studies with 644 participants. We classified articles by running/hiking, cycling, rugby, mixed martial arts (MMA) or other. Eighty-six percent of studies measured a significant increase in a pterin in response to exercise. Changes in pterin concentrations were within 24h of the exercise-stimulus in 79% of studies and 17% measured a change from baseline greater than 48h post-exercise (49% did not measure or report beyond 48h). Neopterin or total neopterin (neopterin+7,8-dihydroneopterin) were the primary pterin measured (28 studies) and they were equally sensitive to exercise regardless of whether the stimulus was running, cycling, rugby, MMA or other.
Neopterin and total neopterin increase in response to exercise-induced stress. Pterins may have limited capacity for monitoring long-term stress beyond 48h but further research is required.
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