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Because of its antiarrhythmic potency and due to the lack of alternatives, amiodarone is often used for antiarrhythmic therapy in patients with ICD or CRT-D systems. To date, robust data on the safety and clinical benefit of amiodarone therapy in these patients are missing OBJECTIVE: This study was designed to assess the periprocedural and post-procedural outcome of combined therapy with beta-blockers plus amiodarone compared to treatment with single beta-blockers in this "real life" cohort of ICD recipients of the German DEVICE registry.4,499 patients who underwent ICD implantation, revision, or upgrade in 49 centers participating in the German DEVICE Registry were enrolled 03/2007-02/2014.Amiodarone had no significant effect on the success of defibrillation testing. Early implantation-associated complications were similar between the groups. One-year overall mortality was, however, significantly higher in the beta-blocker plus amiodarone cohort (adjusted HR 2.09; p0.001). Interestingly, amongst the surviving patients, amiodarone was not associated with a significantly reduced risk of ICD discharges, syncopal events. Further, the occurrence of VT storm or incessant VTs and the number of patients scheduled for intracardiac ablation did not differ among both groups while the rate of rehospitalization was lower in the cohort with sole beta-blockers.While amiodarone has no adverse effect on the success of defibrillation testing, our data suggest an increased all-cause mortality under amiodarone therapy, especially in the subgroups of patients with sinus rhythm or severely reduced left ventricular function. In surviving patients, rates of arrhythmic events were comparable.

Climate change is rapidly warming high latitude and high elevation regions influencing plant community composition. Changes in vegetation composition have motivated the coordination of ecological monitoring networks across the Arctic, including the International Tundra Experiment. We have established a long-term passive warming experiment using open-top chambers, which includes five distinct plant communities (Dry Heath; Tussock Tundra; and Dry, Mesic, and Wet Meadow). We measured changes in plant community composition based on relative abundance differences over 26 years. In addition, relative abundance changes in response to fertilization and warming treatments were analyzed based on a seven-year Community-Level Interaction Program experiment. The communities had distinct soil moisture conditions, leading to community-specific responses of the plant growth forms (deciduous shrubs, evergreen shrubs, forbs, and graminoids). Warming significantly affected growth forms, but the direction of the response was not consistent across the communities. Evidence of shrub expansion was found in nearly all communities, with soil moisture determining whether it was driven by deciduous or evergreen shrubs. Graminoids increased in relative abundance in the Dry Meadow due to warming. Growth form responses to warming are likely mediated by edaphic characteristics of the communities and their interactions with climate.

This study aims to report on the clinical and patient-reported outcomes in patients undergoing atrial fibrillation (AF) ablation with moderate or severe complications.The prospective, multicentre German Ablation Registry of patients undergoing catheter ablation was studied to compare outcomes of patients with moderate or severe complications (group I) and patients without or minor complications (group II).A total of 3865 patients (group I = 158, group II = 3707) were included in this analysis. Procedural success (92.4% vs 96.1%, p = 0.019) was lower and arrhythmia recurrence before discharge significantly higher in group I (15.8% vs 6.5%, p 0.001). Hospital stays were longer in in group I (6 days vs 3 days, p 0.001). The in-hospital rate of death, myocardial infarction (MI) or stroke was 6.4% in group I. Age was an independent predictor of in-hospital complications (HR1.43, 95% CI 1.18-1.72). In the 1-year follow-up, the composite outcome of death, MI, stroke, or major bleeding (8.5% vs 1.5%, p 0.001) was significantly higher in group I. The majority of patients were still feeling safe during treatment regardless of occurred complications (88.4% vs 94.0%, p = 0.14) and would choose the same centre again in most cases (90.7% vs 92.9%, p = 0.59). Patients reported no or improved symptoms in both cohorts (77.3% vs 78.6%) without significant differences.Cardiovascular events in patients with severe complications are more common but patient satisfaction and symptomatic relief are high and comparable to those without complications.

A major goal in the search for extraterrestrial life is the detection of liquid water on the surface of exoplanets. On terrestrial planets, volcanic outgassing is a significant source of atmospheric and surface water and a major contributor to the long-term evolution of the atmosphere. The rate of volcanism depends on the interior evolution and on numerous feedback processes between atmosphere and interior, which continuously shape atmospheric composition, pressure, and temperature. We present the results of a comprehensive 1D model of the coupled evolution of the interior and atmosphere of rocky exoplanets that combines central feedback processes between these two reservoirs. We carried out more than \num{280000} simulations over a wide range of mantle redox states and volatile content, planetary masses, interior structures and orbital distances in order to robustly assess the emergence, accumulation and preservation of surface water on rocky planets. To establish a conservative baseline of which types of planets can outgas and sustain water on their surface, we focus here on stagnant-lid planets. We find that only a narrow range of the mantle redox state around the iron-w\"ustite buffer allows the formation of atmospheres that lead to long-term habitable conditions. At oxidizing conditions similar to those of the Earth's mantle, most stagnant-lid planets end up in a hothouse regime akin to Venus due to strong \ce{CO2} outgassing. At more reducing conditions, the amount of outgassed greenhouse gases is often too low to keep surface water from freezing. In addition, Mercury-like planets with large metallic cores are able to sustain habitable conditions at an extended range of orbital distances as a result of lower volcanic activity., Comment: 23 pages, 18 figures, accepted for publication in Astronomy & Astrophysics

Sympatric adaptive phenotypic divergence should be underlain by genomic differentiation between sub-populations. When divergence drives similar patterns of phenotypic and ecological variation within species we expect evolution to draw on common allelic variation. We investigated divergence histories and genomic signatures of adaptive divergence between benthic and pelagic morphs of Icelandic Arctic charr. Divergence histories for each of four populations were reconstructed using coalescent modelling and 14,187 single nucleotide polymorphisms. Sympatric divergence with continuous gene flow was supported in two populations while allopatric divergence with secondary contact was supported in one population; we could not differentiate between demographic models in the fourth population. We detected parallel patterns of phenotypic divergence along benthic-pelagic evolutionary trajectories among populations. Patterns of genomic differentiation between benthic and pelagic morphs were characterized by outlier loci in many narrow peaks of differentiation throughout the genome, which may reflect the eroding effects of gene flow on nearby neutral loci. We then used genome-wide association analyses to relate both phenotypic (body shape and size) and ecological (carbon and nitrogen stable isotopes) variation to patterns of genomic differentiation. Many peaks of genomic differentiation were associated with phenotypic and ecological variation in the three highly divergent populations, suggesting a genomic basis for adaptive divergence. We detected little evidence for a parallel genomic basis of differentiation as most regions and outlier loci were not shared among populations. Our results show that adaptive divergence can have varied genomic consequences in populations with relatively recent common origins, similar divergence histories, and parallel phenotypic divergence.

Funding Acknowledgements Type of funding sources: None. Background Programmed ventricular stimulation may be considered for risk stratification of sudden cardiac death. The history of syncope is an independent predictor for appropriate device discharge and sudden cardiac death, but data remain sparse among different populations. Purpose The aim of this study was to assess the prognostic value of previous syncope and inducible ventricular arrhythmia (VA) during programmed stimulation in patients undergoing defibrillator implantation. Methods Among 4,196 patients enrolled in the prospective, multi-centre German DEVICE Registry undergoing defibrillator implantation, patients with syncope and inducible VA (n=285, 6.8%) vs. those with a secondary preventive indication (n=1,885, 45.2%) serving as a control group were studied. In an additional analysis of patients with inducible VA, those with vs. without (n=86, 2.1%) previous syncope were compared. Results Patients with syncope and inducible VA (64.9±14.4 years, 81.1% male) presented less often with congestive heart failure (15.1% vs. 29.1%; P Conclusions Patients with a history of syncope and inducible VA present less frequently with most cardiac comorbidities resulting in better outcomes following defibrillator implantation than patients with a secondary preventive indication. Nevertheless, patients with inducible VA experience a relevant number of post-procedural adverse events not differing between those with vs. without syncope, which underlines the importance of VA inducibility, potentially irrespective of the history of syncope.

Funding Acknowledgements Type of funding sources: None. Background/Introduction Catheter ablation of symptomatic atrial fibrillation (AF) is an established treatment option. Cryoballoon (CBA) and radiofrequency ablation (RFA) have demonstrated comparable high rates of freedom from atrial arrhythmia recurrence. Comparative data on quality of life (QoL) outcomes with respect to the used energy source are scarce. Purpose To compare QoL changes following CBA versus RFA in symptomatic AF. Methods This is a sub-analysis of the prospective multi-center FREEZE Cohort Study (ClinicalTrials.gov, NCT01360008). QoL changes were assessed by EQ-5D-3L self-report questionnaire and by EHRA symptom score at baseline and follow-up. Five dimensions of QoL were compared between the treatment groups: "mobility", "self-care", "usual activities", "pain/discomfort", and "anxiety/depression". Changes of Visual Analogue Scale (EQ-VAS) and general physical condition (GPC) were assessed. Results From 2011 to 2016, a total of N=4,189 patients were included, and N=2,110 (50.4%) - CBA N=1,590 (75%) and RFA N=529 (25%) - completed all QoL questionnaires. Differences between groups were observed. Mean age was 61.4±10.4 (CBA) and 63.2±10.4 (RFA) years (p/= II in 89.7% versus 92.9% of patients in CBA and RFA group at baseline (p At follow-up, atrial arrhythmia recurrence was documented in 45.0% and 56.9% of patients in the CBA and RFA group (p/= II was documented in 23.7% and 32.0% of patients (p Significant differences in QoL dimensions from baseline to follow-up were observed between the groups (see Figure): Patients in the CBA group showed a statistically significant greater improvement for the QoL dimension "pain/discomfort", „general physical condition", EQ-VAS, and EQ-5D-Score as compared to the RFA group (all p Conclusion Catheter ablation in symptomatic AF was associated with a significant improvement of AF symptoms, "pain/discomfort", "anxiety/depression", and GPC in both groups. However, there was only a small benefit of ablation on „mobility", „self-care", and „usual activities". Interestingly, in the CBA group patients reported significantly more frequently amelioration of "pain/discomfort" and „general physical condition". This finding was confirmed by a higher EQ-VAS and EQ-5D-Score as well as a higher level of satisfaction in the CBA group, and might be related to the lower recurrence rate.

Funding Acknowledgements Type of funding sources: Other. Main funding source(s): The DEVICE registries were financed by “Stiftung Institut für Herzinfarktforschung (IHF)”, with additional support by grants from Biotronik, Medtronic, and St. Jude Medical. Background Experimental data and early clinical trials suggested that amiodarone may alter the defibrillation threshold of ICD systems. However, because of its potent antiarrhythmic effect and lack of alternatives, amiodarone is frequently used for antiarrhythmic therapy in ICD or CRT-D patients, leading to the question of whether ICD testing with ventricular fibrillation induction should be repeated in these patients after starting amiodarone. Objective This study was designed to assess the impact of amiodarone therapy on the success of ventricular fibrillation induction tests in this "real life" cohort of ICD recipients of the German DEVICE registry. Methods 3,680 patients who underwent ICD implantation, revision, or upgrade in 49 centers participating in the German DEVICE Registry were enrolled 03/2007-02/2014. Results Intraoperative defibrillation testing was conducted in 2,705 patients receiving only beta-blockes as antiarrhythmic therapy and in 422 patients under betablocker plus amiodarone therapy. With regard to ineffective defibrillation tests, no difference could be described between the two groups (0.6% vs. 0.5%; p=0.77). In a similar fashion 488 patients receiving beta-blockers only and 65 patients under beta-blocker plus amiodarone therapy who were scheduled for postoperative defibrillation testing showed comparable rates of ineffective testing (14.5% vs. 15.4%; p=0.86). Conclusions Based on 3,127 intraoperative and 553 postoperative defibrillator testings, our study failed to show a significant association of amiodarone therapy and ineffective defibrillator testings in this "real life" cohort of ICD recipients of the German DEVICE registry. Our results thus underline that, apart from special situations such as right-sided implantation, HCM, extravenous ICD systems, etc., it might not be necessary to perform a DFT test after the start of an amiodarone therapy in ICD patients.

Funding Acknowledgements Type of funding sources: None. Background Implantable cardioverter defibrillators (ICDs) and cardiac resynchronization therapy (CRT) are widely used in patients with life-threatening cardiac arrhythmias or systolic heart failure. Real world data regarding gender impact on ICD and CRT implantation and outcome is scarce. Purpose To investigate the impact of gender-specific differences on cardiac devices implantation and outcomes. Methods The German DEVICE registry is a nationwide prospective multicentre database of ICD and CRT devices implantation. A total of 5451 patients were prospectively enrolled in 44 centres between March 2007 and February 2014 and were monitored for a median of 17 months. Results A minority of patients in this registry was female 1050 (19.3%; mean age 64.4 ± 14.6 years; median BMI 27.2) while men represented 80.7% (mean age 65.5 ± 12.4 years; median BMI 26.8). Female patients were less likely to have a LVEF 55% (13.3% vs. 6.6%; p Similar rates of primary and secondary prevention indication were noted for female and male patients (p=0.9717). There was no difference between the groups regarding the need of postprocedural system revision (p=0.7884), as well as in-hospital death, cardiac death, or sudden cardiac death. The non-fatal complications during follow-up were comparable for men and women. Moreover, similar rates and durations of all cause and cardiac rehospitalizations were found (p=0.1644 and p= 0.1816). Conclusion In this real-life patient cohort only a minority of female patients were enrolled. Female patients were more likely to undergo CRT implantation and had a higher risk for acute major periprocedural complications and in-hospital complications but a lower overall mortality in 1-year follow-up. However, the differences in baseline characteristics should be considered as potential confounders.

Terrestrial gravimetry allows for integrative measurements of mass changes associated with water storage variations in all storage compartments above and below the Earth surface. Superconducting gravimeters (SGs) currently are the most precise instruments for continuous monitoring of gravity change. Their footprint typically covers a radius of about 1 km around the instrument, with most of the signal originating from within the first 100 meters. We installed a SG (iGrav033) in a mixed pine-beech-oak forest in the TERENO observatory in the lowlands of north-eastern Germany. It is housed in a small field enclosure with less than 1 m2 base area, on top of a stable concrete pillar. Complementary hydro-meteorological monitoring data are available at the site, including a weather station, a groundwater monitoring well, clusters of soil moisture sensors along deep soil profiles, interception measurements and near-surface soil moisture from Cosmic Ray Neutron Sensing. For quantification and correction of the long-term instrumental SG drift, repeated measurements with an FG5 absolute gravimeter were carried out. The gravity residual time series (gravity measurements reduced to the local hydrological effect) covers a sequence of years with below average precipitation, from 2018 to 2022. We show the gravity-based water storage variations in the forested landscape throughout this period, indicating that storage depletion during summer in most years is not fully recovered by the subsequent wetter winter periods. The amplitudes of gravity-based water storage variations tend to exceed those observed by soil moisture sensors in the top meters of the soil and of groundwater. This indicates the value of terrestrial gravimetry in revealing dynamics of the deeper unsaturated zone water storage.

The long-term evolution of the atmospheres or rocky planets depends on several different factors, including (but not limited to) volcanic outgassing by partial melting of the rocky interior. Uprising melt may contain different assembladges of volatiles (CHONS) depending on the general mantle composition and redox state, the local volatile inventory, as well as melting depth. All these factors are assumed to vary for mobile-lid planets with an active surface recycling mechanism (e.g. plate tectonics or convective mobile resurfacing) when compared to stagnant-lid planets. In addition, the strength of volcanic activity varies for stagnant-lid and mobile-lid planets, with stronger activity and hence volcanic outgassing expected for the latter case. Atmospheres with pressures above Earth values also severely influence which volatiles will be further outgassed into the atmosphere depending on the solubility of the individual gas species. The outgassing fluxes therefore strongly depend on the evolution of the atmosphere, including atmosphere losses to space or by condensation or weathering. Ultimately, different atmospheric compositions will evolve for planets with low-pressure atmospheres (i.e. low-mass planets, planets without an active surface mobilization process, or planets with efficient atmosphere sinks/losses) and high-pressure atmospheres.Our studies allow us to predict ranges of likely atmospheric properties depending on planet mass and the surface mobility regime, that can then be compared to observations (i.e. with JWST or in the more distant future with the proposed LIFE mission).

Supplementary Data from Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent, and Selective Covalent Oral Inhibitor of KRASG12C

Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors.Significance:JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155.See related video: https://vimeo.com/720726054This article is highlighted in the In This Issue feature, p. 1397

PDF - 60K, Linear correlation observed between tumor growth inhibition (% T/C) or tumor regression and the fraction of time over the in vivo S473P-Akt IC80 in different cancer cell line-derived tumor xenografts implanted in nude mice (represented as dots) and nude rats (represented as triangles) following NVP-BYL719 treatment (R2=0.77, p

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Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials. Mol Cancer Ther; 13(5); 1117–29. ©2014 AACR.

PDF file, 300KB, A. Upon infection with viral particles obtained from the pBabe-puro-myr-Akt retroviral vectors, pools were selected upon addition of puromycin and maintained in culture for several weeks. Extracts from pools were generated for the indicated time of culture and analyzed by Western-blot for the expression of the exogenously expressed HA-tagged form of the dominant active myr-Akt protein. B. 2.5x106 MCF7-BP (upper panel) or MCF7-myr-Akt (bottom panel) were seeded in 10 cm dishes and exposed for 1 h to the indicated compounds at the indicated concentrations (in nM). Cells were then lyzed and cell extracts were analyzed by Western-blot for pathway inhibition as revealed by S473P-Akt levels.

The pan-phosphoinositide 3-kinase (PI3K) inhibitor BKM120 was found, at high concentrations, to cause cell death in various cellular systems, irrespective of their level of PI3K addiction. Transcriptional and biochemical profiling studies were used to identify the origin of these unexpected and apparently PI3K-independent effects. At 5- to 10-fold, the concentration needed to half-maximally inhibit PI3K signaling. BKM120 treatment caused changes in expression of mitotic genes and the induction of a robust G2–M arrest. Tubulin polymerization assays and nuclear magnetic resonance-binding studies revealed that BKM120 inhibited microtubule dynamics upon direct binding to tubulin. To assess the contribution of this off-target activity vis-à-vis the antitumor activity of BKM120 in PI3K-dependent tumors, we used a mechanistic PI3K-α–dependent model. We observed that, in vivo, daily treatment of mice with doses of BKM120 up to 40 mg/kg led to tumor regressions with no increase in the mitotic index. Thus, strong antitumor activity can be achieved in PI3K-dependent models at exposures that are below those necessary to engage the off-target activity. In comparison, the clinical data indicate that it is unlikely that BKM120 will achieve exposures sufficient to significantly engage the off-target activity at tolerated doses and schedules. However, in preclinical settings, the consequences of the off-target activity start to manifest themselves at concentrations above 1 μmol/L in vitro and doses above 50 mg/kg in efficacy studies using subcutaneous tumor–bearing mice. Hence, careful concentration and dose range selection is required to ensure that any observation can be correctly attributed to BKM120 inhibition of PI3K. Mol Cancer Ther; 11(8); 1747–57. ©2012 AACR.

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Schematic illustration of MEKi-induced MAPK pathway feedback activation, SHP099 dose-dependent reduction of p-MEK induction by MEKi, and MEKi-induced p-AKT levels.

Evaluation of the in vitro synergy in anti-proliferation and the MAPK pathway suppression of MEK and SHP2, PI3K, or RTK inhibitors.

PDF file, 53KB, A and B. 5x105 A2058 cells were seeded in 10 cm dishes and incubated for 24 h either with increasing amounts of BKM120 (A) or with 5 �M of either GDC-0941 (B, top panel) or BEZ235 (B, bottom panel). Cells were then fixed, prepared as described for quantification of the population in the different phases of the cell cycle by fluorescence-activated cell sorting. G1, S and G2/M distribution for control untreated cells are described in the mean text and in Figure 4A. The activities of BKM120 on the cell cycle were plotted along to the inhibitory effects on pAkt levels (A).

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PDF - 129K, Legends for Supplementary Figures 1 through 4 and Supplementary Tables 1 through 7. Supplementary Table 1. Determination of NVP-BYL719 in vitro effects on human protein kinases. Supplementary Table 2. Activity profile of NVP-BYL719 in the Invitrogen Kinase panel. Supplementary Table 3. Activity profile of NVP-BYL719 in the Ambit Kinase panel. Supplementary Table 4. NVP-BYL719 Kd (nmol/L) determination in the Ambit Kinase panel for the hits found to be inhibited >90%. Supplementary Table 5. NVP-BYL719 anti-tumor effects in diverse cancer cell line-derived xenograft models. Supplementary Table 6. CCLE cell lines responsive to NVP-BYL719. Supplementary Table 7 Anti-tumor effect of NVP-BYL719 in patient-derived xenograft models carrying PIK3CA genetic alterations.

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PDF file, 107KB, A. 2.5x106 Rat1-myr-p110a (left) or U87MG cells were seeded in 10 cm dishes and exposed for 6h either to BKM120 (5 �M) or to the DMSO control. Cells were then lyzed and cell extracts were analyzed by Western-blot for PI3K pathway inhibition and G2/M markers as revealed by S473P-Akt (upper panel) and Ser10P-Histone H3 (bottom panel) levels, respectively. B. U87MG tumor bearing mice were treated orally, once per day for 6 days with BKM120 at the indicated doses. Tumor volumes were callipered and plotted. ns: not significant.

FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with the MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTK) are also feedback-activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using an SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS-mutant cancer cells, to a less extent in those harboring the G13D variant, and involves several RTKs, including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS, at least for the G12C, G12D, and G12V variants, and wild-type KRAS can also contribute significantly to the feedback activation. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS-mutant cancer cell proliferation in vitro and in vivo. These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic.

PDF file, 113KB, A. Effects of Paclitaxel and Nocodazole on Tubulin polymerization. Tubulin was mixed with either Paclitaxel (10 �M), Nocodazole (10 �M) or the DMSO control in the presence of GTP. The polymerization of monomeric tubulin into microtubule was started by transferring the reaction tubes from 4{degree sign}C to 37{degree sign}C, and monitored by the increase in absorbance (λ=340 nM) over a period of 60 min. B. Competition experiments of NVP-BKM120 with colchicine and podophyllotoxin by NMR spectroscopy. T1ρ relaxation of BKM120 in the presence of tubulin (50-fold excess of compound) remains unchanged after adding podophyllotoxin or colchicine, as emphasized by the drawn arrows. The spectra of the three compounds are shown in three colors at the bottom. C. Structures of GDC0941, BEZ235, Nocodazole and BKM120.

mRNA levels (TPM, transcripts per million reads in RNAseq) of SHP2, RTKs, and their ligands in all cell lines tested.

PDF - 71K, Rat1-myr-p110alpha (blue), beta (green) or delta (red) cells were treated with increasing concentrations of NVP-BYL719 for 30 minutes. Levels of S473P-Akt in cell extracts were quantified by Reverse Phase Protein Array as described in (18) and plotted as percentage of untreated control cells. The graph illustrates n=3 independent experiments. IC50s (plus/minus) SD and IC80s (plus/minus) SD of n=3 independent experiments are reported..

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PDF file, 71KB, The data are scaled by the positive control (1�M MG132) and the negative control (DMSO). The percentage of maximum activity (Amax) is represented in function of the crossing point (concentration in �M at 50% of MG132 activity). Each data point represents a cell line; the vertical and horizontal lines represent, respectively, the median of the crossing point values (1.33�M) and the median of the Amax values (-90.06%), of BKM120 across all cell lines. The populations of cell lines least responding to GDC-0941, among which some are sensitive to BKM120, are highlighted in green.

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Quantification of p-MEK and tubulin levels and percentages of p-MEK reduction in the MEKi combination group normalized to tubulin in all cell lines tested.

PDF file, 653KB, MDA-MB231 (left) and U87MG (right) cells grown on coverslips were treated for 24 h either with BKM120 (5 μM) or Nocodazole (100 nM) and the effects of compound treatment on microtubule dynamics and G2/M arrest was monitored by immuno-fluorescence staining of alpha-tubulin (microtubules), gamma tubulin (centrosomes) and DAPI (DNA). Pictures were taken with a 100X objective.

Validation of HRAS/NRAS double knock-out clones and the contribution of mutant KRAS to the feedback activation in NCI-H358 and MIA PaCa-2.

A list of KRAS mutant cell lines in the study with lineages, zygosity determined by RNAseq, and percentages of p-MEK reduction by 5 micromolar SHP099 in the MEKi combination group.

PDF file, 2791KB. A2058 (upper panels), MDA-MB231 (lower panels) and U87MG cells grown on coverslips were treated for 6 h, 6 h with a subsequent 18 h washout period or 24 h with either 5 μM BKM120 or 100 nM Nocodazole and the effects of compound treatment on microtubule dynamics and G2/M arrest was monitored by immuno-fluorescence staining of alpha-tubulin. Pictures were taken with a 40X objective.

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Changes in phospho-SHP2 levels following MEKi treatment in studied KRAS mutant lines.

PDF - 92K, NVP-BYL719 does not inhibit mTOR and PIKKs involved in DNA damage-repair processes. A. TSC1 -/- MEFs cells were grown in a 96-well format and treated for 1 h with increased concentrations of RAD001 or NVP-BYL719 (from 0.5 nmol/L to 10 ?mol/L in 1 third dilution steps) and immediately fixed. S235/236P-RPS6 levels were measured and IC50 determined with the Excel module XLfit. Background (no primary Ab incubated); BL, Baseline. B: TSC1 -/- MEFs cells were treated with increasing concentrations of NVP-BYL719 as indicated or RAD001 at 500 nmol/L or an equivalent DMSO concentration for 30 minutes. Levels of S235/236P-RPS6 and total RPS6 in protein- normalized lysates were detected by Western blot analyzis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL. C: 24 h post seeding, A549 cells were treated at the same time with Actinomycin D (Act D) at a concentration of 5 ?mol/L (an agent used to induce DNA damage), and with increasing concentrations of NVP-BYL719 as indicated or with the vehicle control (DMSO) for 1 h. Levels of S15P-p53 and tubulin in protein-normalized lysates were detected by Western blot analysis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL. D: 24 h post seeding, U2OS cells were pre-treated for 1 h with increased concentrations of NVP-BYL719 or KU55933 a specific small molecular mass inhibitor of ATM (Supplementary reference 1) at a concentration of 10 ?mol/L or with the vehicle control (DMSO). The cells were then irradiated with 15 Gy and re-incubated at 37 degrees C for 1 h and then lysed. Levels of S1981P-ATM in protein- normalized lysates were detected by Western blot analysis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL.

Evaluation of the in vivo combination benefit and the MAPK pathway suppression of MEK and SHP2 inhibitors in MIA PaCa-2 xenograft and a colon cancer PDX model.

PDF - 37K, NVP-BYL719 was tested at 10 different concentrations (from 0.5 nmol/L to 10 μmol/L in 1 third dilution steps) against CLK2 (A) and LRRK2 (B). SelectScreenTM Kinase Profiling Service uses XLfit from IDBS to determine the IC50 values (IC50 CLK2=621 nmol/L; IC50 LRRK2=3'220 nmol/L). The dose response curve is curve fit to model number 205 (sigmoidal dose-response model).

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PDF file, 57K, Ratio of plasma PlGF or cNRP1 to baseline for each subject at all time points evaluated (Days 14, 21) from the Phase Ia study.

Table S2. Baseline Characteristics in the Tumor SPARC-Evaluable Population