Combined CDK4/6 and ERK1/2 Inhibition Enhances Antitumor Activity in NF1-Associated Plexiform Neurofibroma.

PURPOSE: Plexiform neurofibromas (PNF) are peripheral nerve sheath tumors that cause significant morbidity in persons with neurofibromatosis type 1 (NF1), yet treatment options remain limited. To identify novel therapeutic targets for PNF, we applied an integrated multi-omic approach to quantitatively profile kinome enrichment in a mouse model that has predicted therapeutic responses in clinical trials for NF1-associated PNF with high fidelity. EXPERIMENTAL DESIGN: Utilizing RNA sequencing combined with chemical proteomic profiling of the functionally enriched kinome using multiplexed inhibitor beads coupled with mass spectrometry, we identified molecular signatures predictive of response to CDK4/6 and RAS/MAPK pathway inhibition in PNF. Informed by these results, we evaluated the efficacy of the CDK4/6 inhibitor, abemaciclib, and the ERK1/2 inhibitor, LY3214996, alone and in combination in reducing PNF tumor burden in Nf1flox/flox;PostnCre mice. RESULTS: Converging signatures of CDK4/6 and RAS/MAPK pathway activation were identified within the transcriptome and kinome that were conserved in both murine and human PNF. We observed robust additivity of the CDK4/6 inhibitor, abemaciclib, in combination with the ERK1/2 inhibitor, LY3214996, in murine and human NF1(Nf1) mutant Schwann cells. Consistent with these findings, the combination of abemaciclib (CDK4/6i) and LY3214996 (ERK1/2i) synergized to suppress molecular signatures of MAPK activation and exhibited enhanced antitumor activity in Nf1flox/flox;PostnCre mice in vivo. CONCLUSIONS: These findings provide rationale for the clinical translation of CDK4/6 inhibitors alone and in combination with therapies targeting the RAS/MAPK pathway for the treatment of PNF and other peripheral nerve sheath tumors in persons with NF1.

Effects of Tigecycline Combined with Azithromycin Against Biofilms of Multidrug-Resistant Stenotrophomonas maltophilia Isolates from a Patient in China.

PURPOSE: Our aim was to investigate in vitro biofilm formation by S. maltophilia and the effects of antibacterial agents used to prevent biofilm formation. METHODS: Two trimethoprim/sulfamethoxazole-resistant S. maltophilia strains were isolated from the pleural effusion of a patient with cancer. The minimum inhibitory concentrations (MICs) of amikacin, azithromycin, cefoperazone/sulbactam, and tigecycline were determined. The checkerboard method was used to determine the fractional inhibitory concentration indices (FICIs). A crystal violet biofilm assay and confocal laser scanning microscopy (CLSM) were used to observe biofilm formation. In vitro effects of azithromycin combined with tigecycline on biofilms of S. maltophilia strains were tested. RESULTS: The two S. maltophilia isolates were confirmed to produce strong biofilms. Crystal violet biofilm assay and CLSM analysis of S. maltophilia biofilm were in the initial adhesive stage after 2 h incubation. Biofilm was in the exponential phase of growth at 12 h and reached maximal growth at 36-48 h. Compared with tigecycline or azithromycin alone, the combination of tigecycline and azithromycin increased the inhibiting effect S. maltophilia biofilm biomass after incubation for 12 h. Compared with the control group, in almost all strains treated with tigecycline and azithromycin, the biofilm was significantly suppressed significance (P<0.001). We found that 2x MIC azithromycin combined with 1x MIC tigecycline had the best inhibiting effect against the biofilm, the biofilm inhibition rates of three strains were all over 60%, the biofilm thickness was inhibited from 36.00 ± 4.00 µm to 8.00 µm, from 40.00 µm to 6.67± 2.31 µm, and from 32.00 µm to 13.33 ± 2.31 µm in SMA1, SMA2 and ATCC17666, respectively. CONCLUSION: Azithromycin combined with tigecycline inhibited biofilm formation by S. maltophilia. Our study provides an experimental basis for a possible optimal treatment strategy for S. maltophilia biofilm-related infections.

Retrospective analysis of risk factors of postoperative nausea and vomiting in patients undergoing ambulatory strabismus surgery via general anaesthesia.

BACKGROUND AND AIMS: Postoperative nausea and vomiting (PONV), one of the common complications following strabismus surgery, would delay the ambulatory discharge time. The aim of this retrospective study was to determine the risk factors of PONV in patients undergoing ambulatory strabismus surgery under general anaesthesia, with the treatments of dexamethasone and 5-HT3 antagonist combination. METHODS: We reviewed 721 consecutive patients (12-60 years old) undergoing ambulatory strabismus surgery under general anaesthesia at an academic eye centre between December 2016 and January 2019. Patients received prophylactic treatment of dexamethasone and 5-HT3 antagonist combination during anaesthesia induction, and PONV was evaluated during the early recovery period before discharge. RESULTS: The dexamethasone and 5-HT3 antagonist combination effectively reduced the incidence of PONV (3.05%, 22/721), and the patients who experienced PONV had statistically prolonged phase II recovery time as compared those who did not (P = 0.006). The sum of the extraocular muscles manipulated and the use of nalbuphine (vs flurbiprofen axetil) were the independent risk factors for PONV (P < 0.05). CONCLUSION: The sum of the extraocular muscles manipulated and the use of nalbuphine are potentially modifiable risk factors for PONV after strabismus surgery with the treatments of dexamethasone and 5-HT3 antagonist combination.

Desflurane anesthesia compared with total intravenous anesthesia on anesthesia-controlled operating room time in ambulatory surgery following strabotomy: a randomized controlled study.

BACKGROUND: Ophthalmic ambulatory surgery is preferred to be performed under general anesthesia either by total intravenous anesthesia (TIVA) or by inhalational anesthesia to increase the patient comfort. However, anesthesia-controlled time (ACT) can cause increased non-operative operating room (OR) time which may adversely affect the ORs efficiency. This study was aimed to compare the ACT of desflurane with that of propofol-remifentanil in strabismus ambulatory surgery. METHODS: From November 2016 to December 2017, a total of 200 strabismus patients (aged 18-60 years old, and scheduled for elective ambulatory surgery at Zhongshan Ophthalmic Center) were randomly assigned to receive either propofol-based TIVA (group TIVA) or desflurane anesthesia (group DES) for maintenance of anesthesia. The primary outcome was the extubation time. Secondary outcomes included surgical time, anesthetic time, OR exit time, and Phase I and II recovery time. The intraoperative incidences of hypotension, bradycardia and oculocardiac reflex (OCR), and the incidences of any post-operative complications were recorded. Mann-Whitney U test and Chi-square or Fisher exact tests were used to compare the two groups. RESULTS: We found that the extubation time (5.5 [3.9-7.0] vs. 9.7 [8.5-11.4] min, P < 0.001) and the incidence of prolonged time to extubation (0 vs. 6%, P = 0.029) in the DES group were significantly decreased compared with those in the TIVA group. The patients in the DES group displayed shorter OR exit time as compared with that in the TIVA group (7.3 [5.5-8.7] vs. 10.8 [9.3-12.3] min, P < 0.001). The patients using desflurane exhibited more stable hemodynamics during surgery than the patients using propofol-based TIVA, as demonstrated by lower incidences of hypotension (1% vs. 22%, P < 0.001), bradycardia (2% vs. 13%, P = 0.002), and OCR (17% vs. 44%, P < 0.001). CONCLUSION: DES enhanced the ophthalmic OR efficiency by reducing the extubation time and OR exit time, and provided more stable hemodynamics intra-operatively than TIVA in patients undergoing strabismus ambulatory surgery. TRIAL REGISTRATION: ClinicalTrials.gov, No. NCT02922660; https://clinicaltrials.gov/ct2/show/NCT02922660?id=NCT02922660&draw=2&rank=1.

ERK Inhibitor LY3214996 Targets ERK Pathway-Driven Cancers: A Therapeutic Approach Toward Precision Medicine.

The ERK pathway is critical in oncogenesis; aberrations in components of this pathway are common in approximately 30% of human cancers. ERK1/2 (ERK) regulates cell proliferation, differentiation, and survival and is the terminal node of the pathway. BRAF- and MEK-targeted therapies are effective in BRAF V600E/K metastatic melanoma and lung cancers; however, responses are short-lived due to emergence of resistance. Reactivation of ERK signaling is central to the mechanisms of acquired resistance. Therefore, ERK inhibition provides an opportunity to overcome resistance and leads to improved efficacy. In addition, KRAS-mutant cancers remain an unmet medical need in which ERK inhibitors may provide treatment options alone or in combination with other agents. Here, we report identification and activity of LY3214996, a potent, selective, ATP-competitive ERK inhibitor. LY3214996 treatment inhibited the pharmacodynamic biomarker, phospho-p90RSK1, in cells and tumors, and correlated with LY3214996 exposures and antitumor activities. In in vitro cell proliferation assays, sensitivity to LY3214996 correlated with ERK pathway aberrations. LY3214996 showed dose-dependent tumor growth inhibition and regression in xenograft models harboring ERK pathway alterations. Importantly, more than 50% target inhibition for up to 8 to 16 hours was sufficient for significant tumor growth inhibition as single agent in BRAF- and KRAS-mutant models. LY3214996 also exhibited synergistic combination benefit with a pan-RAF inhibitor in a KRAS-mutant colorectal cancer xenograft model. Furthermore, LY3214996 demonstrated antitumor activity in BRAF-mutant models with acquired resistance in vitro and in vivo. Based on these preclinical data, LY3214996 has advanced to an ongoing phase I clinical trial (NCT02857270).

Cricoid-mental distance-based versus weight-based criteria for size selection of classic laryngeal mask airway in adults: a randomized controlled study.

The optimal size selection of laryngeal mask airway (LMA) based on body weight is not always applicable. This study was prospectively conducted to evaluate the efficacy of cricoid-mental distance-based method versus weight-based method in optimal size selection of LMA in adults. Seventy-four patients (aged from 18 to 65) undergoing ophthalmic surgery were randomly assigned into cricoid-mental (CM) distance-based group or weight-based group to select appropriate size of LMA. The primary outcome was oropharyngeal leak pressure (OLP). Secondary outcomes included overall insertion success rate, number of insertion attempts, time to successful insertion, ease of insertion, score of fiber-optic view, peak inspiratory pressure during mechanical ventilation and postoperative pharyngolaryngeal morbidity. The OLP was significantly higher in CM distance-based group than that in weight-based group (19.38 ± 3.52 vs. 17.50 ± 3.18, P = 0.022). The successful placement at the first attempt in CM distance-based group was dramatically increased as compared with weight-based group (89.2% vs. 62.2%, P = 0.005). The overall success rate of LMA insertion in CM distance-based group was slightly increased in comparison with the weight-based group (100% vs. 91.9%, P = 0.240). There were no significant differences in score of fiber-optic view and postoperative pharyngolaryngeal morbidity between both groups (all P > 0.05). CM distance-based criteria is an alternative choice for optimizing size selection of classic LMA in adults.

Sedation effects of intranasal dexmedetomidine delivered as sprays versus drops on pediatric response to venous cannulation.

OBJECTIVES: Mucosal atomization device (MAD) was designed to increase the bioavailability of intranasal medications by facilitating absorption, the present study aimed to evaluate and compare the sedation effects of intranasal dexmedetomidine delivered as drops versus sprays on pediatric responses to intravenous cannulation. METHODS: One hundred and six pediatric patients (aged from 2 to 5years) scheduled for elective ophthalmic surgery were intranasally received a dose of 2µg/kg in 20µl/kg of dexmedetomidine for sedation to reduce response to venous cannulation. The patients were randomized into syringe group and MAD group in which dexmedetomidine was delivered as drops or sprays via syringe or MAD respectively. The primary outcome was the response to peripheral vein cannulation assessed by the FLACC scores (faces, legs, activity, cry and consolability) 30min after intranasal administration of dexmedetomidine. The secondary outcomes included acceptance for intranasal medication, sedation onset time, and needle insertion times and any adverse event at the preoperative holding area. RESULTS: The FLACC scores in MAD group were significantly decreased than that treated by drops (P=0.021). The acceptance for intranasal administration between both groups was comparable (P>0.05), the onset time and the incidences in two and more times of needle insertion did not differ significantly between syringe and MAD groups (all P>0.05). None of patients were required to clinically intervene in heart rates reduction and none suffered respiratory depression after administrations of dexmedetomidine in either group. CONCLUSION: Intranasal dexmedetomidine by sprays offers better sedation effects to reduce responses to venous cannulation than drops.

Efficacy of premedication with intranasal dexmedetomidine on inhalational induction and postoperative emergence agitation in pediatric undergoing cataract surgery with sevoflurane.

STUDY OBJECTIVES: This study aimed to test the hypothesis that premedication with a single dose of intranasal dexmedetomidine (DEX) could not only reduce preoperative anxiety but also minimize the emergence agitation in children undergoing cataract surgery with sevoflurane anesthesia. DESIGN: Single-blinded, randomized, placebo-controlled clinical comparison study. SETTING: Academic medical center. PATIENTS: Ninety American Society of Anesthesiologists physical status 1 and 2 children scheduled for cataract surgery. INTERVENTIONS: Patients were randomized into 3 groups: group D1, group D2, and saline group (group C), in which the children received 1 or 2 µg/kg of intranasal DEX or saline, respectively, and each group comprises 30 patients. MEASUREMENTS: The mask induction score and the incidences of postoperative emergence agitation evaluated by the Pediatric Anesthesia Emergence Delirium scale were assessed. The emergence time, postanesthesia care unit (PACU) stay time, and any adverse events were recorded. MAIN RESULTS: The mask induction scores were significantly higher in the saline group than those in the D1 and D2 groups (P<.001). The incidences of emergence agitation in the D1 and D2 groups were significantly lower than that in the saline group (7/30 in group D1 and 3/30 in group D2 vs 24/30 in group C, P<.001). The emergence time and PACU stay time were comparable among the 3 groups (P>.05). The emergence time and PACU stay time did not differ significantly in DEX-treated groups as compared with the saline group; there were no differences between 1- and 2-µg/kg groups. None of the patients exhibited significant clinical complications. CONCLUSION: Intranasal DEX (1 or 2 µg/kg) dose independently improves the incidences of mask acceptance and prevents the incidences of postoperative emergency agitation mainly from sevoflurane without delaying the emergency time or inducing severe adverse events.

Targeting SHP2 for EGFR inhibitor resistant non-small cell lung carcinoma.

Targeted therapy with inhibitors of epidermal growth factor receptor (EGFR) has produced a noticeable benefit to non-small cell lung cancer (NSCLC) patients whose tumors carry activating mutations (e.g. L858R) in EGFR. Unfortunately, these patients develop drug resistance after treatment, due to acquired secondary gatekeeper mutations in EGFR (e.g. T790M). Given the critical role of SHP2 in growth factor receptor signaling, we sought to determine whether targeting SHP2 could have therapeutic value for EGFR inhibitor resistant NSCLC. We show that SHP2 is required for EGF-stimulated ERK1/2 phosphorylation and proliferation in EGFR inhibitor resistant NSCLC cell line H1975, which harbors the EGFR T790M/L858R double-mutant. We demonstrate that treatment of H1975 cells with II-B08, a specific SHP2 inhibitor, phenocopies the observed growth inhibition and reduced ERK1/2 activation seen in cells treated with SHP2 siRNA. Importantly, we also find that II-B08 exhibits marked anti-tumor activity in H1975 xenograft mice. Finally, we observe that combined inhibition of SHP2 and PI3K impairs both the ERK1/2 and PI3K/AKT signaling axes and produces significantly greater effects on repressing H1975 cell growth than inhibition of either protein individually. Collectively, these results suggest that targeting SHP2 may represent an effective strategy for treatment of EGFR inhibitor resistant NSCLCs.

SHP2 is a target of the immunosuppressant tautomycetin.

SHP2 phosphatase is a positive transducer of growth factor and cytokine signaling. SHP2 is also a bona fide oncogene; gain-of-function SHP2 mutations leading to increased phosphatase activity cause Noonan syndrome, as well as multiple forms of leukemia and solid tumors. We report that tautomycetin (TTN), an immunosuppressor in organ transplantation, and its engineered analog TTN D-1 are potent SHP2 inhibitors. TTN and TTN D-1 block T cell receptor-mediated tyrosine phosphorylation and ERK activation and gain-of-function mutant SHP2-induced hematopoietic progenitor hyperproliferation and monocytic differentiation. Crystal structure of the SHP2⋅TTN D-1 complex reveals that TTN D-1 occupies the SHP2 active site in a manner similar to that of a peptide substrate. Collectively, the data support the notion that SHP2 is a cellular target for TTN and provide a potential mechanism for the immunosuppressive activity of TTN. Moreover, the structure furnishes molecular insights upon which therapeutics targeting SHP2 can be developed on the basis of the TTN scaffold.

Acute doxorubicin cardiotoxicity is associated with p53-induced inhibition of the mammalian target of rapamycin pathway.

BACKGROUND: Doxorubicin is used to treat childhood and adult cancer. Doxorubicin treatment is associated with both acute and chronic cardiotoxicity. The cardiotoxic effects of doxorubicin are cumulative, which limits its chemotherapeutic dose. Free radical generation and p53-dependent apoptosis are thought to contribute to doxorubicin-induced cardiotoxicity. METHODS AND RESULTS: Adult transgenic (MHC-CB7) mice expressing cardiomyocyte-restricted dominant-interfering p53 and their nontransgenic littermates were treated with doxorubicin (20 mg/kg cumulative dose). Nontransgenic mice exhibited reduced left ventricular systolic function (predoxorubicin fractional shortening [FS] 61+/-2%, postdoxorubicin FS 45+/-2%, mean+/-SEM, P<0.008), reduced cardiac mass, and high levels of cardiomyocyte apoptosis 7 days after the initiation of doxorubicin treatment. In contrast, doxorubicin-treated MHC-CB7 mice exhibited normal left ventricular systolic function (predoxorubicin FS 63+/-2%, postdoxorubicin FS 60+/-2%, P>0.008), normal cardiac mass, and low levels of cardiomyocyte apoptosis. Western blot analyses indicated that mTOR (mammalian target of rapamycin) signaling was inhibited in doxorubicin-treated nontransgenic mice but not in doxorubicin-treated MHC-CB7 mice. Accordingly, transgenic mice with cardiomyocyte-restricted, constitutively active mTOR expression (MHC-mTORca) were studied. Left ventricular systolic function (predoxorubicin FS 64+/-2%, postdoxorubicin FS 60+/-3%, P>0.008) and cardiac mass were normal in doxorubicin-treated MHC-mTORca mice, despite levels of cardiomyocyte apoptosis similar to those seen in doxorubicin-treated nontransgenic mice. CONCLUSIONS: These data suggest that doxorubicin treatment induces acute cardiac dysfunction and reduces cardiac mass via p53-dependent inhibition of mTOR signaling and that loss of myocardial mass, and not cardiomyocyte apoptosis, is the major contributor to acute doxorubicin cardiotoxicity.

Changes in 4E-BP1 and p70S6K phosphorylation in skeletal muscle of the ovine fetus after prolonged maternal fasting: effects of insulin and IGF-I.

This study was conducted to investigate fasting-induced alterations in insulin signaling to the regulatory components of the translation machinery. Insulin (890 mIU/h) and IGF-I (40 nM/h) were infused into a chronically catheterized ovine fetus (0.85 gestation) for 7 h following a 5-d maternal fast. Amino acid and glucose concentrations were clamped to minimize the effects of alterations in circulating substrate concentrations. The IGF-I induced increase in 4E-BP1 phosphorylation (percentage in the gamma form) increased from 28% in control to 44% (NS). The insulin-induced increase in 4E-BP1 phosphorylation was more pronounced, and the gamma percentage was 56% on average in the insulin group. The insulin-induced increase in 4E-BP1 phosphorylation was lower than in fed animals and did not result in significant changes in eIF4E.4E-BP1 binding or eIF4E.eIF4G binding. Insulin increased PKB/Akt phosphorylation and p70S6K phosphorylation to a similar extent as in fed animals. We conclude that maternal fasting resulted in reduced insulin sensitivity of 4E-BP1 phosphorylation and eIF4F formation. This reduced insulin-induced 4E-BP1 phosphorylation was not due to a global defect in insulin signaling; the defects underlying the reduced basal phosphorylation and insulin-responsiveness of 4E-BP1 in fasted animals may be in signaling components other than, or downstream of, PKB/Akt. Selective inhibition of downstream components of insulin signaling allows fetuses to adapt to nutritional stress by decreasing the anabolic response to insulin and other growth factors, so that more amino acids can be used as oxidative substrate to compensate for shortage of energy due to reduced glucose supply.

Insulin and IGF-I stimulate the formation of the eukaryotic initiation factor 4F complex and protein synthesis in C2C12 myotubes independent of availability of external amino acids.

The objective of this study was to investigate the effect of insulin and IGF-I on protein synthesis and translation initiation in C2C12 myotubes in nutrient-deprived Dulbecco's phosphate buffered saline (DPBS). The results showed that insulin and IGF-I increased protein synthesis by 62% and 35% respectively in DPBS, and the effect was not affected by rapamycin, but was blocked by LY294002. Insulin and IGF-I stimulated eukaryotic initiation factor 4E (eIF4E) binding protein (4EBP1) phosphorylation in a dose-dependent manner, and the stimulation was independent of availability of external amino acids. Both LY294002 and rapamycin blocked the insulin and IGF-I-induced increases in 4EBP1 phosphorylation. The results also showed that insulin and IGF-I were able to stimulate PKB/Akt phosphorylation, glycogen synthase kinase (GSK) 3beta phosphorylation and mTOR phosphorylation in DPBS. Insulin and IGF-I increased the amount of eIF4G associated with eIF4E in nutrient-deprived C2C12 myotubes. The amount of 4EBP1 associated with eIF4E was decreased after insulin or IGF-I stimulation. We conclude that in C2C12 myotubes, insulin and IGF-I may regulate protein synthesis and translation initiation independent of external amino acid supply via the phosphatidylinositol-3 kinase-PKB/Akt-mTOR pathway.

IGF-I and insulin regulate eIF4F formation by different mechanisms in muscle and liver in the ovine fetus.

The mechanisms by which insulin-like growth factor I (IGF-I) and insulin regulate eukaryotic initiation factor (eIF)4F formation were examined in the ovine fetus. Insulin infusion increased phosphorylation of eIF4E-binding protein (4E-BP1) in muscle and liver. IGF-I infusion did not alter 4E-BP1 phosphorylation in liver. In muscle, IGF-I increased 4E-BP1 phosphorylation by 27%; the percentage in the gamma-form in the IGF-I group was significantly lower than that in the insulin group. In liver, only IGF-I increased eIF4G. Both IGF-I and insulin increased eIF4E. eIF4G binding in muscle, but only insulin decreased the amount of 4E-BP1 associated with eIF4E. In liver, only IGF-I increased eIF4E. eIF4G binding. Insulin increased the phosphorylation of p70 S6 kinase (p70(S6k)) in both muscle and liver and protein kinase B (PKB/Akt) in muscle, two indicative signal proteins in the phosphatidylinositol (PI) 3-kinase pathway. IGF-I increased PKB/Akt phosphorylation in muscle but had no effect on p70(S6k) phosphorylation in muscle or liver. We conclude that insulin and IGF-I modulate eIF4F formation; however, the two hormones have different regulatory mechanisms. Insulin increases phosphorylation of 4E-BP1 and eIF4E. eIF4G binding in muscle, whereas IGF-I regulates eIF4F formation by increasing total eIF4G. Insulin, but not IGF-I, decreased 4E-BP1 content associated with eIF4E. Insulin regulates translation initiation via the PI 3-kinase-p70(S6k) pathway, whereas IGF-I does so mainly via mechanisms independent of the PI 3-kinase-p70(S6k) pathway.