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Purpose: CT900 is a novel small molecule thymidylate synthase inhibitor that binds to α-folate receptor (α-FR) and thus is selectively taken up by α-FR–overexpressing tumors. Patients and Methods: A 3+3 dose escalation design was used. During dose escalation, CT900 doses of 1–6 mg/m2 weekly and 2–12 mg/m2 every 2 weeks (q2Wk) intravenously were evaluated. Patients with high-grade serous ovarian cancer were enrolled in the expansion cohorts. Results: 109 patients were enrolled: 42 patients in the dose escalation and 67 patients in the expansion cohorts. At the dose/schedule of 12 mg/m2/q2Wk (with and without dexamethasone, n = 40), the most common treatment-related adverse events were fatigue, nausea, diarrhea, cough, anemia, and pneumonitis, which were predominantly grade 1 and grade 2. Levels of CT900 more than 600 nmol/L needed for growth inhibition in preclinical models were achieved for >65 hours at a dose of 12 mg/m2. In the expansion cohorts, the overall response rate (ORR), was 14/64 (21.9%). Thirty-eight response-evaluable patients in the expansion cohorts receiving 12 mg/m2/q2Wk had tumor evaluable for quantification of α-FR. Patients with high or medium expression had an objective response rate of 9/25 (36%) compared with 1/13 (7.7%) in patients with negative/very low or low expression of α-FR. Conclusions: The dose of 12 mg/m2/q2Wk was declared the recommended phase II dose/schedule. At this dose/schedule, CT900 exhibited an acceptable side effect profile with clinical benefit in patients with high/medium α-FR expression and warrants further investigation.

Pedigree analysis showed that a large proportion of Leber hereditary optic neuropathy (LHON) family members who carry a mitochondrial risk variant never lose vision. Mitochondrial haplotype appears to be a major factor influencing the risk of vision loss from LHON. Mitochondrial variants, including m.14484TC and m.11778GA, have been added to gene arrays, and thus many patients and research participants are tested for LHON mutations. Analysis of the UK Biobank and Australian cohort studies found more than 1 in 1,000 people in the general population carry either the m.14484TC or the m.11778GA LHON variant. None of the subset of carriers examined had visual acuity at 20/200 or worse, suggesting a very low penetrance of LHON. Haplogroup analysis of m.14484TC carriers showed a high rate of haplogroup U subclades, previously shown to have low penetrance in pedigrees. Penetrance calculations of the general population are lower than pedigree calculations, most likely because of modifier genetic factors. This Matters Arising Response paper addresses the Watson et al. (2022) Matters Arising paper, published concurrently in The American Journal of Human Genetics.

Disease recurrence is common following prostatectomy in patients with localised prostate cancer with high-risk features. Although androgen deprivation therapy increases the rates of organ-confined disease and negative surgical margins, there is no significant benefit on disease recurrence. Multiple lines of evidence suggest that (Fibroblast Growth Factor/Fibroblast Growth Factor Receptor) FGF/FGFR-signalling is important in supporting prostate epithelial cell survival in hostile conditions, including acute androgen deprivation. Given the recent availability of oral FGFR inhibitors, we investigated whether combination therapy could improve tumour response in the neo-adjuvant setting.We conducted an open label phase II study of the combination of erdafitinib (3 months) and androgen deprivation therapy (4 months) in men with localised prostate cancer with high-risk features prior to prostatectomy using a Simon's 2 stage design. The co-primary endpoints were safety and tolerability and pathological response in the prostatectomy specimen. The effect of treatment on residual tumours was explored by global transcriptional profiling with RNA-sequencing.Nine patients were enrolled in the first stage of the trial. The treatment combination was poorly tolerated. Erdafitinib treatment was discontinued early in six patients, three of whom also required dose interruptions/reductions. Androgen deprivation therapy for 4 months was completed in all patients. The most common adverse events were hyperphosphataemia, taste disturbance, dry mouth and nail changes. No patients achieved a complete pathological response, although patients who tolerated erdafitinib for longer had smaller residual tumours, associated with reduced transcriptional signatures of epithelial cell proliferation.Although there was a possible enhanced anti-tumour effect of androgen deprivation therapy in combination with erdafitnib in treatment naïve prostate cancer, the poor tolerability in this patient population prohibits the use of this combination in this setting.

This work is concerned with providing a principled decision process for stopping or tool-changing in a surface finishing process. The decision process is supposed to work for products of non-flat geometry. The solution is based on conducting hypothesis testing on the bearing area curves from two consecutive stages of a surface finishing process. In each stage, the bearing area curves, which are in fact the nonparametric quantile curves representing the surface roughness, are extracted from surface profile measurements at a number of sampling locations on the surface of the products. The hypothesis test of these curves informs the decision makers whether there is a change in surface quality induced by the current finishing action. When such change is detected, the current action is deemed effective and should thus continue, while when no change is detected, the effectiveness of the current action is then called into question, signaling possibly some change in the course of action. Application of the hypothesis testing-based decision procedure to both spherical and flat surfaces demonstrates the effectiveness and benefit of the proposed method and confirms its geometry-agnostic nature., 33 pages, 12 figures

Event sequences are central to the analysis of data in domains that range from biology and health, to logfile analysis and people's everyday behavior. Many visualization tools have been created for such data, but people are error-prone when asked to judge the similarity of event sequences with basic presentation methods. This article describes an experiment that investigates whether local and global alignment techniques improve people's performance when judging sequence similarity. Participants were divided into three groups (basic versus local versus global alignment), and each participant judged the similarity of 180 sets of pseudo-randomly generated sequences. Each set comprised a target, a correct choice and a wrong choice. After training, the global alignment group was more accurate than the local alignment group (98 versus 93 percent correct), with the basic group getting 95 percent correct. Participants' response times were primarily affected by the number of event types, the similarity of sequences (measured by the Levenshtein distance) and the edit types (nine combinations of deletion, insertion and substitution). In summary, global alignment is superior and people's performance could be further improved by choosing alignment parameters that explicitly penalize sequence mismatches.

Introduction. When emergency medical service (EMS) personnel respond to emergencies, the decisions they make can often mean life or death for the patient. This is especially true in the case of advanced airway management. Protocols are set in place to ensure that the least invasive airway management techniques are used initially before more invasive techniques. The purpose of this study was to determine how often EMS personnel followed this protocol, while adequately achieving the goals of appropriate oxygenation and ventilation. Methods. This retrospective chart review was approved by the Institutional Review Board of The University of Kansas Medical Center. The authors reviewed the Wichita/Sedgewick County EMS system for cases in which patients required airway support. We examined de-identified data to determine if invasive methods were applied in sequence. Cohen's kappa coefficient (κ) and immersion-crystallization approach were used to analyze the data. Results. A total of 279 cases were identified in which EMS personnel used advanced airway management techniques. In 90% (n = 251) of cases, less invasive techniques were not used prior to more invasive techniques and in 80% (n = 222) of cases, the more invasive technique was used alone. A dirty airway was the most common reason for the EMS choice using more invasive approaches in achieving the goals of appropriate oxygenation and ventilation. Conclusions. Our data showed that EMS personnel in Sedgwick County/Wichita, Kansas often deviate from the advanced airway management protocols when caring for patients in need of respiratory intervention. Dirty airway was found to be the main reason for using a more invasive approach in achieving the goals of appropriate oxygenation and ventilation. It is important to fully understand reasons why deviations in protocol are occurring to ensure that current protocols, documentation, and training practices are effective in producing the best possible patient outcomes.

Background National audits aim to reduce variations in quality by stimulating quality improvement. However, varying provider engagement with audit data means that this is not being realised. Aim The aim of the study was to develop and evaluate a quality dashboard (i.e. QualDash) to support clinical teams’ and managers’ use of national audit data. Design The study was a realist evaluation and biography of artefacts study. Setting The study involved five NHS acute trusts. Methods and results In phase 1, we developed a theory of national audits through interviews. Data use was supported by data access, audit staff skilled to produce data visualisations, data timeliness and quality, and the importance of perceived metrics. Data were mainly used by clinical teams. Organisational-level staff questioned the legitimacy of national audits. In phase 2, QualDash was co-designed and the QualDash theory was developed. QualDash provides interactive customisable visualisations to enable the exploration of relationships between variables. Locating QualDash on site servers gave users control of data upload frequency. In phase 3, we developed an adoption strategy through focus groups. ‘Champions’, awareness-raising through e-bulletins and demonstrations, and quick reference tools were agreed. In phase 4, we tested the QualDash theory using a mixed-methods evaluation. Constraints on use were metric configurations that did not match users’ expectations, affecting champions’ willingness to promote QualDash, and limited computing resources. Easy customisability supported use. The greatest use was where data use was previously constrained. In these contexts, report preparation time was reduced and efforts to improve data quality were supported, although the interrupted time series analysis did not show improved data quality. Twenty-three questionnaires were returned, revealing positive perceptions of ease of use and usefulness. In phase 5, the feasibility of conducting a cluster randomised controlled trial of QualDash was assessed. Interviews were undertaken to understand how QualDash could be revised to support a region-wide Gold Command. Requirements included multiple real-time data sources and functionality to help to identify priorities. Conclusions Audits seeking to widen engagement may find the following strategies beneficial: involving a range of professional groups in choosing metrics; real-time reporting; presenting ‘headline’ metrics important to organisational-level staff; using routinely collected clinical data to populate data fields; and dashboards that help staff to explore and report audit data. Those designing dashboards may find it beneficial to include the following: ‘at a glance’ visualisation of key metrics; visualisations configured in line with existing visualisations that teams use, with clear labelling; functionality that supports the creation of reports and presentations; the ability to explore relationships between variables and drill down to look at subgroups; and low requirements for computing resources. Organisations introducing a dashboard may find the following strategies beneficial: clinical champion to promote use; testing with real data by audit staff; establishing routines for integrating use into work practices; involving audit staff in adoption activities; and allowing customisation. Limitations The COVID-19 pandemic stopped phase 4 data collection, limiting our ability to further test and refine the QualDash theory. Questionnaire results should be treated with caution because of the small, possibly biased, sample. Control sites for the interrupted time series analysis were not possible because of research and development delays. One intervention site did not submit data. Limited uptake meant that assessing the impact on more measures was not appropriate. Future work The extent to which national audit dashboards are used and the strategies national audits use to encourage uptake, a realist review of the impact of dashboards, and rigorous evaluations of the impact of dashboards and the effectiveness of adoption strategies should be explored. Study registration This study is registered as ISRCTN18289782. Funding This project was funded by the National Institute for Health and Care Research (NIHR) Health and Social Care Delivery Research programme and will be published in full in Health and Social Care Delivery Research; Vol. 10, No. 12. See the NIHR Journals Library website for further project information.

Axenfeld-Rieger syndrome encompasses a group of developmental disorders affecting the anterior chamber structures of the eye, with associated systemic features in some cases. This study aims to compare the difference in anterior segment phenotypes such as those involving the cornea, iris, lens, and anterior chamber angle between cases with disease-causing sequence variations in FOXC1 and PITX2 .This cross-sectional study involved 61 individuals, from 32 families with pathogenic FOXC1 or PITX2 variants, who were registered with the Australian and New Zealand Registry of Advanced Glaucoma.The median age of the cohort was 39 years at the time of last assessment (range 3-85 years; females, 54%). Thirty-two patients had pathogenic variants in the FOXC1 gene, and 29 patients had pathogenic variants in the PITX2 gene. Corneal abnormalities were more common in individuals with FOXC1 variants (18/36, 50%) than those with PITX2 variants (4/25, 16%; P = 0.007). Iris abnormalities such as hypoplasia ( P = 0.008) and pseudopolycoria ( P = 0.001) were more common in individuals with PITX2 variants than those with FOXC1 variants. Glaucoma was present in 72% of participants. Corneal decompensation was positively associated with corneal abnormalities ( P0.001), glaucoma surgery ( P = 0.025), and cataract surgery ( P = 0.002).Corneal abnormalities were more common in individuals with FOXC1 than in those with PITX2 variants and were often associated with early onset glaucoma. These findings highlight that patients with FOXC1 variations require close follow-up and monitoring throughout infancy and into adulthood.

Purpose: To conduct a costing study comparing orthoptist-led with consultant-led clinics screening for optic pathway gliomas (OPGs) in children with neurofibromatosis Type 1 (NF1) attending the Royal Children’s Hospital (RCH), Melbourne. Methods: Patients with NF1 examined in the orthoptist-led NF1 screening clinic and/or consultant-led clinics during the study period were identified. The workflow management software Q-Flow 6® provided data documenting patient’s time spent with the orthoptist, nurse, and ophthalmologist. Time points were converted into minutes and multiplied by the cost-per-minute for each profession. A bottom-up micro-costing approach was used to estimate appointment level costs. Bootstrap simulations with 1000 replications were used to estimate 95% confidence intervals (CIs) for the difference in mean appointment time and cost between clinics. Results: Data for 130 consultant-led clinic appointments and 234 orthoptist-led clinic appointments were extracted for analysis. The mean time per appointment for the consultant-led clinic was 45.11 minutes, and the mean time per appointment for the orthoptist-led clinic was 25.85 minutes. The mean cost per appointment for the consultant-led clinic was A $84.15 (GBP £39.60) compared to the orthoptist-led clinic at A $20.40 (GBP £9.60). This represents a mean reduction of 19.25 minutes per appointment (95% CI, –24.85 to –13.66) and a mean reduction of A $63.75 (GBP £30.00) per appointment (95% CI, (A $-75.40 to $-52.10 [GBP £ -35.48 to £ -24.52]). Conclusion: An orthoptist-led clinic screening for OPGs in patients with NF1 can be a more cost-efficient model of care for ophthalmic screening in this patient group.

Escalation phase dose levels and final recruitment

Preclinical studies have demonstrated synergy between PARP and PI3K/AKT pathway inhibitors in BRCA1 and BRCA2 (BRCA1/2)–deficient and BRCA1/2-proficient tumors. We conducted an investigator-initiated phase I trial utilizing a prospective intrapatient dose- escalation design to assess two schedules of capivasertib (AKT inhibitor) with olaparib (PARP inhibitor) in 64 patients with advanced solid tumors. Dose expansions enrolled germline BRCA1/2-mutant tumors, or BRCA1/2 wild-type cancers harboring somatic DNA damage response (DDR) or PI3K–AKT pathway alterations. The combination was well tolerated. Recommended phase II doses for the two schedules were: olaparib 300 mg twice a day with either capivasertib 400 mg twice a day 4 days on, 3 days off, or capivasertib 640 mg twice a day 2 days on, 5 days off. Pharmacokinetics were dose proportional. Pharmacodynamic studies confirmed phosphorylated (p) GSK3β suppression, increased pERK, and decreased BRCA1 expression. Twenty-five (44.6%) of 56 evaluable patients achieved clinical benefit (RECIST complete response/partial response or stable disease ≥ 4 months), including patients with tumors harboring germline BRCA1/2 mutations and BRCA1/2 wild-type cancers with or without DDR and PI3K–AKT pathway alterations.Significance:In the first trial to combine PARP and AKT inhibitors, a prospective intrapatient dose- escalation design demonstrated safety, tolerability, and pharmacokinetic–pharmacodynamic activity and assessed predictive biomarkers of response/resistance. Antitumor activity was observed in patients harboring tumors with germline BRCA1/2 mutations and BRCA1/2 wild-type cancers with or without somatic DDR and/or PI3K–AKT pathway alterations.This article is highlighted in the In This Issue feature, p. 1426

Supplementary Tables 1-7; Supplementary Figures 1-2; Supplementary Methods

Baseline tissue biomarker analysis

Supplementary Figures 1-4 from Preclinical Pharmacology, Antitumor Activity, and Development of Pharmacodynamic Markers for the Novel, Potent AKT Inhibitor CCT128930

Supplementary Figure Legends and Supplementary Table 1 from Preclinical Pharmacology, Antitumor Activity, and Development of Pharmacodynamic Markers for the Novel, Potent AKT Inhibitor CCT128930

RMH200 panel design

Pharmacokinetics

Supplementary Table from Individualized Prediction of Drug Response and Rational Combination Therapy in NSCLC Using Artificial Intelligence–Enabled Studies of Acute Phosphoproteomic Changes

Progression free survival by baseline ESR1

Circulating tumor DNA dynamics as a surrogate of efficacy

Cyclin-dependent kinase 4/6 (CDK4/6) and PI3K inhibitors synergize in PIK3CA-mutant ER-positive HER2-negative breast cancer models. We conducted a phase Ib trial investigating the safety and efficacy of doublet CDK4/6 inhibitor palbociclib plus selective PI3K inhibitor taselisib in advanced solid tumors, and triplet palbociclib plus taselisib plus fulvestrant in 25 patients with PIK3CA-mutant, ER-positive HER2-negative advanced breast cancer. The triplet therapy response rate in PIK3CA-mutant, ER-positive HER2-negative cancer was 37.5% [95% confidence interval (CI), 18.8–59.4]. Durable disease control was observed in PIK3CA-mutant ER-negative breast cancer and other solid tumors with doublet therapy. Both combinations were well tolerated at pharmacodynamically active doses. In the triplet group, high baseline cyclin E1 expression associated with shorter progression-free survival (PFS; HR = 4.2; 95% CI, 1.3–13.1; P = 0.02). Early circulating tumor DNA (ctDNA) dynamics demonstrated high on-treatment ctDNA association with shorter PFS (HR = 5.2; 95% CI, 1.4–19.4; P = 0.04). Longitudinal plasma ctDNA sequencing provided genomic evolution evidence during triplet therapy.SIGNIFICANCE:The triplet of palbociclib, taselisib, and fulvestrant has promising efficacy in patients with heavily pretreated PIK3CA-mutant ER-positive HER2-negative advanced breast cancer. A subset of patients with PIK3CA-mutant triple-negative breast cancer derived clinical benefit from palbociclib and taselisib doublet, suggesting a potential nonchemotherapy targeted approach for this population.This article is highlighted in the In This Issue feature, p. 1

Somatic mutations in ACVR1 are found in a quarter of children with diffuse intrinsic pontine glioma (DIPG), but there are no ACVR1 inhibitors licensed for the disease. Using an artificial intelligence–based platform to search for approved compounds for ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (Kd = 150 nmol/L) and reduce DIPG cell viability in vitro but has limited ability to cross the blood–brain barrier. In addition to mTOR, everolimus inhibited ABCG2 (BCRP) and ABCB1 (P-gp) transporters and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination was well tolerated in vivo and significantly extended survival and reduced tumor burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Four patients with ACVR1-mutant DIPG were treated with vandetanib plus an mTOR inhibitor, informing the dosing and toxicity profile of this combination for future clinical studies.Significance:Twenty-five percent of patients with the incurable brainstem tumor DIPG harbor somatic activating mutations in ACVR1, but there are no approved drugs targeting the receptor. Using artificial intelligence, we identify and validate, both experimentally and clinically, the novel combination of vandetanib and everolimus in these children based on both signaling and pharmacokinetic synergies.This article is highlighted in the In This Issue feature, p. 275

AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment- and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G1 arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials. Mol Cancer Ther; 10(2); 360–71. ©2010 AACR.

We hypothesize that the study of acute protein perturbation in signal transduction by targeted anticancer drugs can predict drug sensitivity of these agents used as single agents and rational combination therapy. We assayed dynamic changes in 52 phosphoproteins caused by an acute exposure (1 hour) to clinically relevant concentrations of seven targeted anticancer drugs in 35 non–small cell lung cancer (NSCLC) cell lines and 16 samples of NSCLC cells isolated from pleural effusions. We studied drug sensitivities across 35 cell lines and synergy of combinations of all drugs in six cell lines (252 combinations). We developed orthogonal machine-learning approaches to predict drug response and rational combination therapy. Our methods predicted the most and least sensitive quartiles of drug sensitivity with an AUC of 0.79 and 0.78, respectively, whereas predictions based on mutations in three genes commonly known to predict response to the drug studied, for example, EGFR, PIK3CA, and KRAS, did not predict sensitivity (AUC of 0.5 across all quartiles). The machine-learning predictions of combinations that were compared with experimentally generated data showed a bias to the highest quartile of Bliss synergy scores (P = 0.0243). We confirmed feasibility of running such assays on 16 patient samples of freshly isolated NSCLC cells from pleural effusions. We have provided proof of concept for novel methods of using acute ex vivo exposure of cancer cells to targeted anticancer drugs to predict response as single agents or combinations. These approaches could complement current approaches using gene mutations/amplifications/rearrangements as biomarkers and demonstrate the utility of proteomics data to inform treatment selection in the clinic.

Efficacy of doublet palbociclib plus taselesib in advanced solid tumors

Supplementary Figures S1-S3

Supplementary Figure from A Phase I Trial of CT900, a Novel α-Folate Receptor–Mediated Thymidylate Synthase Inhibitor, in Patients with Solid Tumors with Expansion Cohorts in Patients with High-Grade Serous Ovarian Cancer

Supplementary Table from A Phase I Trial of CT900, a Novel α-Folate Receptor–Mediated Thymidylate Synthase Inhibitor, in Patients with Solid Tumors with Expansion Cohorts in Patients with High-Grade Serous Ovarian Cancer

Purpose:CT900 is a novel small molecule thymidylate synthase inhibitor that binds to α-folate receptor (α-FR) and thus is selectively taken up by α-FR–overexpressing tumors.Patients and Methods:A 3+3 dose escalation design was used. During dose escalation, CT900 doses of 1–6 mg/m2 weekly and 2–12 mg/m2 every 2 weeks (q2Wk) intravenously were evaluated. Patients with high-grade serous ovarian cancer were enrolled in the expansion cohorts.Results:109 patients were enrolled: 42 patients in the dose escalation and 67 patients in the expansion cohorts. At the dose/schedule of 12 mg/m2/q2Wk (with and without dexamethasone, n = 40), the most common treatment-related adverse events were fatigue, nausea, diarrhea, cough, anemia, and pneumonitis, which were predominantly grade 1 and grade 2. Levels of CT900 more than 600 nmol/L needed for growth inhibition in preclinical models were achieved for >65 hours at a dose of 12 mg/m2. In the expansion cohorts, the overall response rate (ORR), was 14/64 (21.9%). Thirty-eight response-evaluable patients in the expansion cohorts receiving 12 mg/m2/q2Wk had tumor evaluable for quantification of α-FR. Patients with high or medium expression had an objective response rate of 9/25 (36%) compared with 1/13 (7.7%) in patients with negative/very low or low expression of α-FR.Conclusions:The dose of 12 mg/m2/q2Wk was declared the recommended phase II dose/schedule. At this dose/schedule, CT900 exhibited an acceptable side effect profile with clinical benefit in patients with high/medium α-FR expression and warrants further investigation.

CCR Translation for This Article from Dual Blockade of the PI3K/AKT/mTOR (AZD8055) and RAS/MEK/ERK (AZD6244) Pathways Synergistically Inhibits Rhabdomyosarcoma Cell Growth In Vitro and In Vivo

Demographic Profile

Purpose:AT13148 is an oral AGC kinase inhibitor, which potently inhibits ROCK and AKT kinases. In preclinical models, AT13148 has been shown to have antimetastatic and antiproliferative activity.Patients and Methods:The trial followed a rolling six design during dose escalation. An intrapatient dose escalation arm to evaluate tolerability and a biopsy cohort to study pharmacodynamic effects were later added. AT13148 was administered orally three days a week (Mon–Wed–Fri) in 28-day cycles. Pharmacokinetic profiles were assessed using mass spectrometry and pharmacodynamic studies included quantifying p-GSK3β levels in platelet-rich plasma (PRP) and p-cofilin and p-MLC2 levels in tumor biopsies.Results:Fifty-one patients were treated on study. The safety of 5–300 mg of AT13148 was studied. Further, the doses of 120–180–240 mg were studied in an intrapatient dose escalation cohort. The dose-limiting toxicities included hypotension (300 mg), pneumonitis, and elevated liver enzymes (240 mg), and skin rash (180 mg). The most common side effects were fatigue, nausea, headaches, and hypotension. On the basis of tolerability, 180 mg was considered the maximally tolerated dose. At 180 mg, mean Cmax and AUC were 400 nmol/L and 13,000 nmol/L/hour, respectively. At 180 mg, ≥50% reduction of p-cofilin was observed in 3 of 8 posttreatment biopsies.Conclusions:AT13148 was the first dual potent ROCK-AKT inhibitor to be investigated for the treatment of solid tumors. The narrow therapeutic index and the pharmacokinetic profile led to recommend not developing this compound further. There are significant lessons learned in designing and testing agents that simultaneously inhibit multiple kinases including AGC kinases in cancer.

Supplementary Table & Figure Legends - PDF file 41K, Detailed captions for: Suppl Table S1. Immunohistochemical analysis indicates correlation between pAKT(Ser473),pERK1/2(Thr202/Tyr204) and pS6(Ser235/236) in primary tumours Fig S1. High level expression of p110alpha in two of four ARMS cell lines. Fig S2. PIK3CA KD does not induce cell cycle arrest or apoptosis in the growth inhibited RMS cells. Fig S3. Downregulation of PTEN expression appears to be associated with delayed inhibition of AKT phosphorylation in RH30 cells following p110alpha KD. Fig S4 Increased sensitivity of the RMS-1 PIK3CA KD line to TGX221, and of the RMS-1, RD and RMS-YM KD lines to ZSTK474. Fig S5 Equivalent changes in the biomarkers of MAPK and PI3K activity at 6h in vivo, to those seen in vitro following acute dosing with AZD6244 and AZD8055 alone, and in combination, with recovery to control levels correlating with plasma and tumor clearance of both drugs. Fig S6 Tolerability study of twice daily dosing of 10mk/kg po AZD6244 and AZD8055 indicates reversible weight loss with combination treatment, while PK analysis of tail vein blood spots confirms drug-drug interactions. Fig S7 Dose-finding therapeutic study defines a once daily treatment schedule of AZD6244, 10mg/kg p.o., and AZD8055, 20mg/kg p.o., as well tolerated and active when given in combination

Inclusion/Exclusion Criteria and Pharmacokinetic Analytical Method

Supplementary Methods - PDF file 44K, Method for the preparation and analysis of AZD8055, AZD6244 and BEZ235 by LC-MS/MS Drug-drug interaction assay

Relationship between dose and AUC/Cmax

Purpose: To provide rationale for using phosphoinositide 3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathway inhibitors to treat rhabdomyosarcomas, a major cause of pediatric and adolescent cancer deaths.Experimental Design: The prevalence of PI3K/MAPK pathway activation in rhabdomyosarcoma clinical samples was assessed using immunohistochemistry. Compensatory signaling and cross-talk between PI3K/MAPK pathways was determined in rhabdomyosarcoma cell lines following p110α short hairpin RNA–mediated depletion. Pharmacologic inhibition of reprogrammed signaling in stable p110α knockdown lines was used to determine the target-inhibition profile inducing maximal growth inhibition. The in vitro and in vivo efficacy of inhibitors of TORC1/2 (AZD8055), MEK (AZD6244), and P13K/mTOR (NVP-BEZ235) was evaluated alone and in pairwise combinations.Results: PI3K pathway activation was seen in 82.5% rhabdomyosarcomas with coactivated MAPK in 36% and 46% of alveolar and embryonal subtypes, respectively. p110α knockdown in cell lines over the short and long term was associated with compensatory expression of other p110 isoforms, activation of the MAPK pathway, and cross-talk to reactivate the PI3K pathway. Combinations of PI3K pathway and MAP–ERK kinase (MEK) inhibitors synergistically inhibited cell growth in vitro. Treatment of RD cells with AZD8055 plus AZD6244 blocked reciprocal pathway activation, as evidenced by reduced AKT/ERK/S6 phosphorylation. In vivo, the synergistic effect on growth and changes in pharmacodynamic biomarkers was recapitulated using the AZD8055/AZD6244 combination but not NVP-BEZ235/AZD6244. Pharmacokinetic analysis provided evidence of drug–drug interaction with both combinations.Conclusions: Dual PI3K/MAPK pathway activation and compensatory signaling in both rhabdomyosarcoma subtypes predict a lack of clinical efficacy for single agents targeting either pathway, supporting a therapeutic strategy combining a TORC1/2 with a MEK inhibitor. Clin Cancer Res; 19(21); 5940–51. ©2013 AACR.

Supplementary Table and Figures - PDF file 184K, Suppl Table S1: Immunohistochemical analysis indicates correlation between pAKT(Ser473),pERK1/2(Thr202/Tyr204) and pS6(Ser235/236) in primary tumours Fig S1. High level expression of p110delta in two of four ARMS cell lines. Fig S2. PIK3CA KD does not induce cell cycle arrest or apoptosis in the growth inhibited RMS cells. Fig S3. Downregulation of PTEN expression appears to be associated with delayed inhibition of AKT phosphorylation in RH30 cells following p110� KD. Fig S4 Increased sensitivity of the RMS-1 PIK3CA KD line to TGX221, and of the RMS-1, RD and RMS-YM KD lines to ZSTK474. Fig S5 Equivalent changes in the biomarkers of MAPK and PI3K activity at 6h in vivo, to those seen in vitro following acute dosing with AZD6244 and AZD8055 alone, and in combination, with recovery to control levels correlating with plasma and tumor clearance of both drugs. Fig S6 Tolerability study of twice daily dosing of 10mk/kg po AZD6244 and AZD8055 indicates reversible weight loss with combination treatment, while PK analysis of tail vein blood spots confirms drug-drug interactions. Fig S7 Dose-finding therapeutic study defines a once daily treatment schedule of AZD6244, 10mg/kg p.o., and AZD8055, 20mg/kg p.o., as well tolerated and active when given in combination

Supplementary Figure 5 from A Useful Approach to Identify Novel Small-Molecule Inhibitors of Wnt-Dependent Transcription

Supplementary Figures 1-3 from A Useful Approach to Identify Novel Small-Molecule Inhibitors of Wnt-Dependent Transcription

Supplementary Figure Legends 1-13, Methods from A Useful Approach to Identify Novel Small-Molecule Inhibitors of Wnt-Dependent Transcription

Supplementary Figure 13 from A Useful Approach to Identify Novel Small-Molecule Inhibitors of Wnt-Dependent Transcription

Supplementary Figure 12 from A Useful Approach to Identify Novel Small-Molecule Inhibitors of Wnt-Dependent Transcription

Supplementary Table 1. Median latency, penetration, tumor characteristics for Th-MYCN/Trp53WT/WT, Th-MYCN/Trp53KI/WT and Th-MYCN/Trp53KI/KI mice. Supplementary Table 2. Sequencing results for Trp53 exons 5-9. Supplementary Table 3. KEGG pathway analysis of genes differentially expressed in Th-MYCN/Trp53KI/KI tumors versus Th-MYCN/Trp53WT/WT tumors

Supplementary Figure 1. The p53 pathway is functional in Th-MYCN neuroblastomas. Supplementary Figure 2. Immunohistochemical analysis of Th-MYCN mice. Supplementary Figure 3. Restoration of p53 has no effect on tumor growth rate or penetration in Th-MYCN/Trp53KI/KI mice. Supplementary Figure 4. Th-MYCN/Trp53KI/KI pups display increased percentage of neoplastic islands compared to Th-MYCN/Trp53WT/WT pups. Supplementary Figure 5. Figure S5 Th-MYCN/Trp53WT/WT and Th-MYCN/Trp53KI/K tumors are similar in their basal DNA damage levels.

Supplementary Figure 10 from A Useful Approach to Identify Novel Small-Molecule Inhibitors of Wnt-Dependent Transcription

Supplementary Figures 6-9 from A Useful Approach to Identify Novel Small-Molecule Inhibitors of Wnt-Dependent Transcription