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Mental illness screening instruments are increasingly being administered through online patient portals, making it vital to understand how the design of digital screening technologies could alter screening scores. Given the strong cross-cultural belief in the gender depression disparity, digital screening technologies are at particular risk of triggering stereotype threat, the phenomenon where a reminder of a stereotype impacts task performance. To assess this risk, we investigate if a reminder about the gender depression disparity influences the scores of digitally administered mental screening instruments. In a comprehensive study, we collect data from 440 participants with a mobile application that reminds half of the participants of the gender depression disparity prior to administering depression and anxiety screening instruments. Our statistical analysis evaluates differences in screening scores with t-tests, and determines credible values for difference of means, of standard deviations, and effect size using Bayesian estimation. While the gender depression disparity reminder had no statistically significant impact on men, it did alter the depression screening scores of women and nonbinary participants. Further, prior depression treatment increased the impact of stereotype threat on women. Our research demonstrates that digital screening technologies are subject to stereotype threat and should thus be designed to avoid biasing mental illness screening scores.

Cell-based therapy (CBT) is attracting much attention to treat incurable diseases. In recent years, several clinical trials have been conducted using human pluripotent stem cells (hPSCs), and other potential therapeutic cells. Various private- and government-funded organizations are investing in finding permanent cures for diseases that are difficult or expensive to treat over a lifespan, such as age-related macular degeneration, Parkinson’s disease, or diabetes, etc. Clinical-grade cell manufacturing requiring current good manufacturing practices (cGMP) has therefore become an important issue to make safe and effective CBT products. Current cell production practices are adopted from conventional antibody or protein production in the pharmaceutical industry, wherein cells are used as a vector to produce the desired products. With CBT, however, the “cells are the final products” and sensitive to physico- chemical parameters and storage conditions anywhere between isolation and patient administration. In addition, the manufacturing of cellular products involves multi-stage processing, including cell isolation, genetic modification, PSC derivation, expansion, differentiation, purification, characterization, cryopreservation, etc. Posing a high risk of product contamination, these can be time- and cost- prohibitive due to maintenance of cGMP. The growing demand of CBT needs integrated manufacturing systems that can provide a more simple and cost-effective platform. Here, we discuss the current methods and limitations of CBT, based upon experience with biologics production. We review current cell manufacturing integration, automation and provide an overview of some important considerations and best cGMP practices. Finally, we propose how multi-stage cell processing can be integrated into a single bioreactor, in order to develop streamlined cGMP-compliant cell processing systems.

Abstract Given the current opioid crisis around the world, harm reduction agencies are seeking to help people who use drugs to do so more safely. Many harm reduction agencies are exploring techniques to test illicit drugs to identify and, where possible, quantify their constituents allowing their users to make informed decisions. While these technologies have been used for years in Europe (Nightlife Empowerment & Well-being Implementation Project, Drug Checking Service: Good Practice Standards; Trans European Drugs Information (TEDI) Workgroup, Factsheet on Drug Checking in Europe, 2011; European Monitoring Centre for Drugs and Drug Addiction, An Inventory of On-site Pill-Testing Interventions in the EU: Fact Files, 2001), they are only now starting to be utilized in this context in North America. The goal of this paper is to describe the most common methods for testing illicit substances and then, based on this broad, encompassing review, recommend the most appropriate methods for testing at point of care. Based on our review, the best methods for point-of-care drug testing are handheld infrared spectroscopy, Raman spectroscopy, and ion mobility spectrometry; mass spectrometry is the current gold standard in forensic drug analysis. It would be prudent for agencies or clinics that can obtain the funding to contact the companies who produce these devices to discuss possible usage in a harm reduction setting. Lower tech options, such as spot/color tests and immunoassays, are limited in their use but affordable and easy to use.

Design enhancements are often added to charts, signage, and infographics to help garner attention or communicate a message. Though it is argued that they may also detract user’s focus on the underlying information, prior studies have contributed evidence that visual design enhancements and even simple decorations can improve memory of visual displays. However, there is limited empirical knowledge about how the type of aesthetic enhancements infuences memory, and what informational and data elements are remembered from a visualization. We conducted a user study testing chart types (line, pie, and bar), the presence of color, and whether added enhancements were contextually related to the data topic presented in each chart. We found that enhancements relevant to the data helped in the recall of title and thematic elements, but enhancements did not signifcantly affect recall of specifc data values. This suggests that using relevant enhancements can have a positive effect on memorability of some chart content, but only if the design styles are chosen well to match the information topic. Recall of chart topics for unrelated embellishments was worse than plain, un-enhanced charts, which suggests that visual enhancement can distract or interfere with memorability if the viewer does not understand a meaningful connection between informational topic and design modifcations.

This report studies different properties of a Gaussian cannon. The cannon’s firing velocity is measured for a varying number of steel balls, varying input velocity and different geometries of the cannon. By measuring the attractive force from the magnet on the balls a mapping of the potential energy for the firing process is acquired. It is found that the firing of a Gaussian cannon can be modeled as successive collisions between the magnet and its neighboring balls, and between successive balls. To reach the highest possible ejection speed with a trigger ball that is released from rest, a chained cannon should be used. The optimal number of balls on each individual cannon depends on magnetization energies and coefficients of restitution.

Background: Lisavanbulin (BAL101553) is a small, lipophilic, oral microtubule destabilizer with promising antitumoral activity observed in preclinical glioblastoma (GBM) models., Methods: This multicenter phase 1 study sought to determine the MTD of oral Lisavanbulin in combination with standard RT (60 Gy/30 fractions) but without temozolomide in patients with newly diagnosed MGMT promoter unmethylated GBM (uGBM). Dose escalation followed a modified 3 + 3 design. Secondary objectives included estimation of OS and PFS and pharmacokinetic analysis., Results: Twenty-six patients with uGBM (median age, 63 years, 42.3% male, 61.5% with gross total resection, median Karnofsky performance status 80) were enrolled; 2 tumors had an IDH1 mutation. Predefined dose levels of Lisavanbulin, administered daily concomitantly with RT, were: 4 mg (5 pts), 6 mg (5 pts), 8 mg (7 pts), 12 mg (5 pts), and 15 mg (4 pts). The initial starting dose was 8 mg. Due to grade 4 aseptic meningoencephalitis in the first patient, the dose was decreased to 4 mg. Dose escalation resumed and continued to 15 mg with dose-limiting toxicities of grade 2 confusion and memory impairment observed at 12 mg. Avanbulin exposures increased in a relatively dose-proportional manner with increasing oral dose of Lisavanbulin from 4 to 15 mg., Conclusions: Lisavanbulin in combination with RT was considered safe up to the highest predefined oral dose level of 15 mg daily., Competing Interests: M.H. has served in a consulting capacity with Novartis, Servier, AnHeart, Bayer, and he serves as a member of a data safety monitoring board with Parexel and Advarra. R.E.S. has served in a consulting capacity with Novocure, Monteris Medical, Alexion, and Springworks. M.E., T.K., H.L., and K.L. are employees of Basilea Pharmaceutica International Ltd, Allschwil. H.L.: Basilea stock/shareholder., (© The Author(s) 2024. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Background: Threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1) are common essential kinases that collaborate in activating the spindle assembly checkpoint (SAC) at the kinetochore, ensuring appropriate chromosome alignment and segregation prior to mitotic exit. Targeting of either TTK or PLK1 has been clinically evaluated in cancer patients; however, dual inhibitors have not yet been pursued. Here we present the in vitro and in vivo characterization of a first in class, dual TTK/PLK1 inhibitor (BAL0891)., Methods: Mechanism of action studies utilized biochemical kinase and proteomics-based target-engagement assays. Cellular end-point assays included immunoblot- and flow cytometry-based cell cycle analyses and SAC integrity evaluation using immunoprecipitation and immunofluorescence approaches. Anticancer activity was assessed in vitro using cell growth assays and efficacy was evaluated, alone and in combination with paclitaxel and carboplatin, using mouse models of triple negative breast cancer (TNBC)., Results: BAL0891 elicits a prolonged effect on TTK, with a transient activity on PLK1. This unique profile potentiates SAC disruption, forcing tumor cells to aberrantly exit mitosis with faster kinetics than observed with a TTK-specific inhibitor. Broad anti-proliferative activity was demonstrated across solid tumor cell lines in vitro . Moreover, intermittent intravenous single-agent BAL0891 treatment of the MDA-MB-231 mouse model of TNBC induced profound tumor regressions associated with prolonged TTK and transient PLK1 in-tumor target occupancy. Furthermore, differential tumor responses across a panel of thirteen TNBC patient-derived xenograft models indicated profound anticancer activity in a subset (~40%). Using a flexible dosing approach, pathologically confirmed cures were observed in combination with paclitaxel, whereas synergy with carboplatin was schedule dependent., Conclusions: Dual TTK/PLK1 inhibition represents a novel approach for the treatment of human cancer, including TNBC patients, with a potential for potent anticancer activity and a favorable therapeutic index. Moreover, combination approaches may provide an avenue to expand responsive patient populations., Competing Interests: EZ, NF-G, FB, PM, KB, AG, MR, MS and HL were all previous employees of Basilea Pharmaceutica International Ltd, Allschwil. AB, KL, LK and LB are current employees of Basilea Pharmaceutica International Ltd, Allschwil. MS, AB, KL, LK and HL are also Basilea Pharmaceutica International Ltd, Allschwil, stockholders. HL is also a consultant for SillaJen Inc. NW-S, HR and GZ are employees of Oncolines BV and previous Netherlands Translational Research Center NTRC B.V. employees. JM, D-VP and RB are employees of Crossfire Oncology BV formerly Netherlands Translational Research Center NTRC B.V. The studies presented in this manuscript were funded by Basilea Pharmaceutica International Ltd, Allschwil. The funder contributed to study design, as well as the collection, analysis, and interpretation of the results., (Copyright © 2024 Zanini, Forster-Gross, Bachmann, Brüngger, McSheehy, Litherland, Burger, Groner, Roceri, Bury, Stieger, Willemsen-Seegers, de Man, Vu-Pham, van Riel, Zaman, Buijsman, Kellenberger and Lane.)

Derazantinib (DZB) is an inhibitor of the fibroblast growth factor receptors 1-3 (FGFRi) with similar potency against colony-stimulating factor receptor-1 (CSF1R), a protein important in the recruitment and function of tumor-associated macrophages. DZB inhibited pCSF1R in the macrophage cell line RAW264.7, and tumor cells GDM-1 and DEL, and had the same potency in HeLa cells transiently over-expressing FGFR2. DZB exhibited similar potency against pCSF1R expressed by isolated murine macrophages, but as in the cell lines, specific FGFRi were without significant CSF1R activity. DZB inhibited growth of three tumor xenograft models with reported expression or amplification of CSF1R, whereas the specific FGFRi, pemigatinib, had no efficacy. In the FGFR-driven syngeneic breast tumor-model, 4T1, DZB was highly efficacious causing tumor stasis. A murine PD-L1 antibody was without efficacy in this model, but combined with DZB, increased efficacy against the primary tumor and further reduced liver, spine and lung metastases. Immunohistochemistry of primary 4T1 tumors showed that the combination favored an antitumor immune infiltrate by strongly increasing cytotoxic T, natural killer and T-helper cells. Similar modulation of the tumor microenvironment was observed in an FGFR-insensitive syngeneic bladder model, MBT-2. These data confirm CSF1R as an important oncology target for DZB and provide mechanistic insight for the ongoing clinical trials, in which DZB is combined with the PD-L1 antibody, atezolizumab., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Derazantinib (DZB) is an inhibitor of fibroblast growth factor receptors 1-3 (FGFR1-3), with additional activity against colony-stimulating-factor-1 receptor (CSF1R). We have profiled the activity of DZB in gastric cancer (GC) as monotherapy and combined with paclitaxel, and explored means of stratifying patients for treatment. The antiproliferative potency of DZB in vitro was quantified in 90 tumor cell lines and shown to correlate significantly with FGFR expression (<0.01) but not with FGFR DNA copy-number (CN) or FGFR mutations. In four GC cell lines in vitro , little or no synergy was observed with paclitaxel. In athymic nude mice, bearing cell-line derived xenografts (CDX) or patient-derived xenograft (PDX) GC models, DZB efficacy correlated highly significantly with FGFR gene expression ( r2 = 0.58; P = 0.0003; n = 18), but not FGFR mutations or DNA-CN. In FGFR-driven GC models, DZB had comparable efficacy to three other FGFR inhibitors and was more efficacious than paclitaxel. DZB had dose-dependent plasma pharmacokinetics but showed low brain penetration at all doses. GC models (one CDX and six PDX) were tested for sensitivity to the combination of DZB and paclitaxel and characterized by immunohistochemistry. The combination showed synergy (5) or additivity (2), and no antagonism, with synergy significantly associated ( P < 0.05) with higher levels of M2-type macrophages. The association of strong efficacy of the combination in vivo with M2 macrophages, which are known to express CSF1R, and the absence of synergy in vitro is consistent with the tumor microenvironment also being a factor in DZB efficacy and suggests additional means by which DZB could be stratified for cancer treatment in the clinic., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Glioblastoma (GBM) is an incurable disease with few approved therapeutic interventions. Radiation therapy (RT) and temozolomide (TMZ) remain the standards of care. The efficacy and optimal deployment schedule of the orally bioavailable small-molecule tumor checkpoint controller lisavanbulin alone, and in combination with, standards of care were assessed using a panel of IDH-wildtype GBM patient-derived xenografts., Methods: Mice bearing intracranial tumors received lisavanbulin +/-RT +/-TMZ and followed for survival. Lisavanbulin concentrations in plasma and brain were determined by liquid chromatography with tandem mass spectrometry, while flow cytometry was used for cell cycle analysis., Results: Lisavanbulin monotherapy showed significant benefit (P < .01) in 9 of 14 PDXs tested (median survival extension 9%-84%) and brain-to-plasma ratios of 1.3 and 1.6 at 2- and 6-hours postdose, respectively, validating previous data suggesting significant exposure in the brain. Prolonged lisavanbulin dosing from RT start until moribund was required for maximal benefit (GBM6: median survival lisavanbulin/RT 90 vs. RT alone 69 days, P = .0001; GBM150: lisavanbulin/RT 143 days vs. RT alone 73 days, P = .06). Similar observations were seen with RT/TMZ combinations (GBM39: RT/TMZ/lisavanbulin 502 days vs. RT/TMZ 249 days, P = .0001; GBM26: RT/TMZ/lisavanbulin 172 days vs. RT/TMZ 121 days, P = .04). Immunohistochemical analyses showed a significant increase in phospho-histone H3 with lisavanbulin treatment (P = .01)., Conclusions: Lisavanbulin demonstrated excellent brain penetration, significant extension of survival alone or in RT or RT/TMZ combinations, and was associated with mitotic arrest. These data provide a strong clinical rationale for testing lisavanbulin in combination with RT or RT/TMZ in GBM patients., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glioblastoma is a highly malignant brain tumor with no curative treatment options, and immune checkpoint blockade has not yet shown major impact. We hypothesized that drugs targeting mitosis might affect the tumor microenvironment and sensitize cancer cells to immunotherapy. We used 2 glioblastoma mouse models with different immunogenicity profiles, GL261 and SB28, to test the efficacy of antineoplastic and immunotherapy combinations. The spindle assembly checkpoint activator BAL101553 (lisavanbulin), agonistic anti-CD40 antibody, and double immune checkpoint blockade (anti-programmed cell death 1 and anti-cytotoxic T lymphocyte-associated protein 4; anti-PD-1 and anti-CTLA-4) were evaluated individually or in combination for treating orthotopic GL261 and SB28 tumors. Genomic and immunological analyses were used to predict and interpret therapy responsiveness. BAL101553 monotherapy increased survival in immune checkpoint blockade-resistant SB28 glioblastoma tumors and synergized with anti-CD40 antibody, in a T cell-independent manner. In contrast, the more immunogenic and highly mutated GL261 model responded best to anti-PD-1 and anti-CTLA-4 therapy and more modestly to BAL101553 and anti-CD40 combination. Our results show that BAL101553 is a promising therapeutic agent for glioblastoma and could synergize with innate immune stimulation. Overall, these data strongly support immune profiling of glioblastoma patients and preclinical testing of combination therapies with appropriate models for particular patient groups.

The association between specific HLA alleles and risk for posttransplantation diabetes (PTDM) in a contemporary and multiethnic kidney transplant recipient cohort is not clear., Methods: In this single-center analysis, data were retrospectively analyzed for 1560 nondiabetic kidney transplant recipients at a single center between 2007 and 2018, with median follow-up of 33 mo (interquartile range 8-73). HLA typing methodology was by DNA analysis and reported at the resolution required for the national allocation scheme. Diagnosis of PTDM was aligned with International Consensus recommendations., Results: PTDM developed in 231 kidney transplant recipients. Exploring 99 HLA alleles, the presence of Cw12, B52, B38, B58, DQ4, A80, and DR13 and the absence of DQ3 and DR04 were associated with significant increases in PTDM risk. In a multivariable Cox regression model, adjusting for other clinical risk factors for PTDM, the presence of Cw12 (hazard ratio [HR], 1.57; 95% CI, 1.08-2.27; P  = 0.017) and DQ4 (HR, 1.78; 95% CI, 1.07-2.96; P  = 0.026) were found to be independent risk factors for PTDM. There was also evidence that the presence of B58 increases PTDM risk within the subgroup of recipients of White ethnicity (HR, 5.01; 95% CI, 2.20-11.42; P  < 0.001)., Conclusion: Our data suggest that specific HLA alleles can be associated with PTDM risk, which can be used pretransplantation for PTDM risk stratification. However, association is not causality, and this work requires replication and further investigation to understand underlying biological mechanisms., Competing Interests: The authors declare no funding or conflicts of interest., (Copyright © 2021 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

Background: BAL101553 (lisavanbulin), the lysine prodrug of BAL27862 (avanbulin), exhibits broad anti-proliferative activity in human cancer models refractory to clinically relevant microtubule-targeting agents., Methods: This two-part, open-label, phase 1/2a study aimed to determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of 2-h infusion of BAL101553 in adults with advanced or recurrent solid tumours. The MTD was determined using a modified accelerated titration design in phase I. Patients received BAL101553 at the MTD and at lower doses in the phase 2a expansion to characterise safety and efficacy and to determine the recommended phase 2 dose (RP2D)., Results: Seventy-three patients received BAL101553 at doses of 15-80 mg/m 2 (phase 1, n = 24; phase 2a, n = 49). The MTD was 60 mg/m 2 ; DLTs observed at doses ≥60 mg/m 2 were reversible Grade 2-3 gait disturbance with Grade 2 peripheral sensory neuropathy. In phase 2a, asymptomatic myocardial injury was observed at doses ≥45 mg/m 2 . The RP2D for 2-h intravenous infusion was 30 mg/m 2 . The overall disease control rate was 26.3% in the efficacy population., Conclusions: The RP2D for 2-h infusion of BAL101553 was well tolerated. Dose-limiting neurological and myocardial side effects were consistent with the agent's vascular-disrupting properties., Clinical Trial Registration: EudraCT: 2010-024237-23.

Glioblastoma (GBM) are aggressive brain tumors with limited treatment options. Cancer stem-like cells (CSLCs) contribute to GBM invasiveness, representing promising targets. BAL101553, a prodrug of BAL27862, is a novel small molecule tubulin-binding agent, promoting tumor cell death through spindle assembly checkpoint activation, which is currently in Phase 1/2a in advanced solid tumor patients including GBM. This study aimed to evaluate long-term daily oral BAL101553 treatment of mice orthotopically grafted with GBM CSLCs (GBM6) according to EB1 expression-level, and to decipher its mechanism of action on GBM stem cells. Oral treatment with BAL101553 for 100 days provoked a large EB1 expression level-dependent survival benefit, together with a decrease in tumor growth and brain invasion. Formation of vascular structures by the fluorescent GBM6-GFP-sh0 cells, mimicking endothelial vascular networks, was observed in the brains of control grafted mice. Following BAL101553 treatment, vessels were no longer detectable, suggesting inhibition of the endothelial trans-differentiation of GBM stem cells. In vitro , BAL27862 treatment resulted in a switch to the endothelial-like phenotype of GBM6 towards an astrocytic phenotype. Moreover, the drug inhibited secretion of VEGF, thus preventing normal endothelial cell migration activated by CSLCs. The decrease in VEGF secretion was confirmed in a human GBM explant following drug treatment. Altogether, our data first confirm the potential of EB1 expression as a response-predictive biomarker of BAL101553 in GBM we previously published and add new insights in BAL101553 long-term action by counteracting CSLCs mediated tumor angiogenesis. Our results strongly support BAL101553 clinical studies in GBM patients., Competing Interests: CONFLICTS OF INTEREST HAL and FB are employed by Basilea Pharmaceutic International AG and own Basilea stocks/options. Basilea fundings partially contributed to experimental costs., (Copyright: © 2019 Bergès et al.)

Highly glycolytic cancer cells prevent intracellular acidification by excreting the glycolytic end-products lactate and H + via the monocarboxylate transporters 1 (MCT1) and 4 (MCT4). We report that syrosingopine, an anti-hypertensive drug, is a dual MCT1 and MCT4 inhibitor (with 60-fold higher potency on MCT4) that prevents lactate and H + efflux. Syrosingopine elicits synthetic lethality with metformin, an inhibitor of mitochondrial NADH dehydrogenase. NAD+, required for the ATP-generating steps of glycolysis, is regenerated from NADH by mitochondrial NADH dehydrogenase or lactate dehydrogenase. Syrosingopine treatment leads to high intracellular lactate levels and thereby end-product inhibition of lactate dehydrogenase. The loss of NAD+ regeneration capacity due to combined metformin and syrosingopine treatment results in glycolytic blockade, leading to ATP depletion and cell death. Accordingly, ATP levels can be partly restored by exogenously provided NAD+, the NAD precursor nicotinamide mononucleotide (NMN), or vitamin K2. Thus, pharmacological inhibition of MCT1 and MCT4 combined with metformin treatment is a potential cancer therapy., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Resistance to microtubule targeting agents (MTA) represents a major drawback in successful cancer therapy with MTAs. Here we investigated the combined treatment modality of the novel MTA BAL101553 in combination with radiotherapy in paclitaxel and epothilone-resistant tumor models., Material and Methods: Multiple regimens of BAL101553, or its active moiety BAL27862 for in vitro experiments, were probed in combination with radiotherapy in P-glycoprotein-overexpressing, human colon adenocarcinoma cells (SW480) and in tubulin-mutated human NSCLC cells (A549EpoB40) and tumors thereof., Results: BAL27862 reduced the proliferative activity of SW480 and A549EpoB40 tumor cells with similar potency as in A549 wildtype cells. Combined treatment of BAL27862 with ionizing radiation in vitro resulted in an additive reduction of clonogenicity. Moreover, treatment of paclitaxel- and epothilone-resistant tumors with fractionated irradiation and different regimens of BAL101553 (a single i.v. bolus vs. oral daily) suppressed tumor growth and resulted in an extended additive tumor growth delay with strong reduction of tumor proliferation and mean tumor vessel density., Conclusions: BAL101553 is a promising alternative in taxane- and epothilone-refractory tumors as part of a combined treatment modality with ionizing radiation. Its potent antitumor effect is not only tumor cell-directed but also targets the tumor microenvironment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We report that the anticancer activity of the widely used diabetic drug metformin is strongly potentiated by syrosingopine. Synthetic lethality elicited by combining the two drugs is synergistic and specific to transformed cells. This effect is unrelated to syrosingopine's known role as an inhibitor of the vesicular monoamine transporters. Syrosingopine binds to the glycolytic enzyme α-enolase in vitro, and the expression of the γ-enolase isoform correlates with nonresponsiveness to the drug combination. Syrosingopine sensitized cancer cells to metformin and its more potent derivative phenformin far below the individual toxic threshold of each compound. Thus, combining syrosingopine and codrugs is a promising therapeutic strategy for clinical application for the treatment of cancer.

Glioblastoma patients have limited treatment options. Cancer stem-like cells (CSLC) contribute to glioblastoma invasiveness and repopulation; hence, they represent promising targets for novel therapies. BAL101553 is a prodrug of BAL27862, a novel microtubule-destabilizing agent inhibiting tumor cell proliferation through activation of the spindle assembly checkpoint, which is currently in phase I/II clinical development. Broad anticancer activity has been demonstrated against human cancer models, including tumors refractory to conventional treatments. We have shown that overexpression of microtubule + end-binding 1-protein (EB1) correlates with glioblastoma progression and poor survival. Here, we show that BAL27862 inhibits the growth of two glioblastoma CSLCs. As EB1 is overexpressed in the CSLC line GBM6, which displays a high tumorigenicity and infiltrative pattern of migration in vivo, we investigated drug activity on GBM6 according to EB1 expression. BAL27862 inhibited migration and colony formation at subcytotoxic concentrations in EB1-expressing control cells (GBM6-sh0) but only at cytotoxic concentrations in EB1-downregulated (GBM-shE1) cells. Three administrations of BAL101553 were sufficient to provoke an EB1-dependent survival benefit in tumor-bearing mice. Patterns of invasion and quantification of tumor cells in brain demonstrated that GBM6-sh0 cells were more invasive than GBM6-shEB1 cells, and that the antiproliferative and anti-invasive effects of BAL101553 were more potent in mice bearing control tumors than in EB1-downregulated tumors. This was associated with inhibition of stem cell properties in the GBM6-sh0 model. Finally, BAL27862 triggered astrocytic differentiation of GBM6 in an EB1-dependent manner. These results support the potential of BAL101553 for glioblastoma treatment, with EB1 expression as a predictive biomarker of response. Mol Cancer Ther; 15(11); 2740-9. ©2016 AACR., (©2016 American Association for Cancer Research.)

The valves of oysters act as a physical barrier between tissues and the external environment, thereby protecting the oyster from environmental stress and predation. To better understand differences in shell properties and predation susceptibilities of two physiologically and morphologically similar oysters, Crassostrea virginica and Crassostrea ariakensis, we quantified and compared two mechanical properties of shells: hardness (resistance to irreversible deformation; GPa) and compressive strength (force necessary to produce a crack; N). We found no differences in the hardness values between foliated layers (innermost and outermost foliated layers), age class (C. virginica: 1, 4, 6, 9 years; C. ariakensis: 4, 6 years), or species. This suggests that the foliated layers have similar properties and are likely composed of the same material. The compressive force required to break wet and dry shells was also not different. However, the shells of both six- and nine-year-old C. virginica withstood higher compressive force than C. virginica shells aged either one or four, and the shells of C. ariakensis at both ages studied (4- and 6-years-old). Differences in ability to withstand compressive force are likely explained by differences in thickness and density between age classes and species. Further, we compared the compressive strength of differing ages of these two species to the crushing force of common oyster predators in the Chesapeake Bay. By studying the physical properties of shells, this work may contribute to a better understanding of the mechanical defenses of oysters as well as of their predation vulnerabilities.

Introduction: Strategies to improve the efficacy of endocrine agents in breast cancer (BC) therapy and to delay the onset of resistance include concomitant targeting of the estrogen receptor alpha (ER) and the mammalian target of rapamycin complex 1 (mTORC1), which regulate cell-cycle progression and are supported by recent clinical results., Methods: BC cell lines expressing aromatase (AROM) and modeling endocrine-sensitive (MCF7-AROM1) and human epidermal growth factor receptor 2 (HER2)-dependent de novo resistant disease (BT474-AROM3) and long-term estrogen-deprived (LTED) MCF7 cells that had acquired resistance associated with HER2 overexpression were treated in vitro and as subcutaneous xenografts with everolimus (RAD001-mTORC1 inhibitor), in combination with tamoxifen or letrozole. End points included proliferation, cell-cycle arrest, cell signaling, and effects on ER-mediated transactivation., Results: Everolimus caused a concentration-dependent decrease in proliferation in all cell lines, which was associated with reductions in S6 phosphorylation. Everolimus plus letrozole or tamoxifen enhanced the antiproliferative effect and G1-accumulation compared with monotherapy, as well as increased phosphorylation (Ser10) and nuclear accumulation of p27 and pronounced dephosphorylation of Rb. Sensitivity was greatest to everolimus in the LTED cells but was reduced by added estrogen. Increased pAKT occurred in all circumstances with everolimus and, in the BT474 and LTED cells, was associated with increased pHER3. Decreased ER transactivation suggested that the effectiveness of everolimus might be partly related to interrupting cross-talk between growth-factor signaling and ER. In MCF7-AROM1 xenografts, letrozole plus everolimus showed a trend toward enhanced tumor regression, versus the single agents. In BT474-AROM3 xenografts, everolimus alone was equally effective at reducing tumor volume as were the combination therapies., Conclusions: The results provide mechanistic support for recent positive clinical data on the combination of everolimus and endocrine therapy, as well as data on potential routes of escape via enhanced HER2/3 signaling. This merits investigation for further improvements in treatment efficacy.

Introduction: The critical role of increased activity of mammalian target of rapamycin (mTOR) in the pathophysiology of multiple diseases is well established. Inhibition of the mTOR pathway may block disease progression and improve patient outcomes. Everolimus, an mTOR inhibitor, began in clinical development as part of a regimen (Certican, Zortress) for prevention of organ transplant rejection and is now an approved oncology agent., Areas Covered: The objective of this review is to discuss the history of key findings and innovative cancer research undertaken to successfully develop everolimus as an oncology therapy (Afinitor) now approved for patients with advanced renal cell carcinoma (RCC) and for subependymal giant cell astrocytomas (SEGAs) associated with tuberous sclerosis. In addition, data for the use of everolimus in the treatment of other cancers and rare diseases are also discussed. A PubMed search of English articles without time restrictions was conducted using the search terms 'everolimus or rapamycin' and 'cancer'. Bibliographies of retrieved articles were manually searched for additional relevant articles. Major cancer congresses were also searched., Expert Opinion: The clinical efficacy of everolimus alone and in combination with other agents has been observed in recently completed Phase II-III studies in a wide spectrum of tumors, including RCC, neuroendocrine tumors, tuberous sclerosis complex, SEGAs and angiomyolipomas, lymphoma and gastric, breast and hepatocellular cancers. These findings emphasize the importance of mTOR in diverse cancers and rare diseases and underscore the potential role for everolimus as an effective agent in multiple indications.

Purpose: Everolimus (RAD001, Afinitor) is an mTORC1 pathway inhibitor, and vatalanib (PTK/ZK) is a pan VEGF-R tyrosine kinase inhibitor (TKI). These two drugs have been shown to have overlapping but also distinct anti-angiogenic effects. Consequently, we investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of their combination in vivo., Methods: Murine melanoma B16/BL6 cells were grown orthotopically in BL6/C57 mice by injection into the derma of both ears to create a primary tumour which metastasized rapidly to the cervical lymph nodes. Mice were treated daily p.o. with PTK/ZK (100 mg/kg) or everolimus (1 mg/kg) or their combination, and anti-tumour efficacy (PD) assessed. In the same model, plasma PK of everolimus was measured following single doses of the monotherapy or combination schedules., Results: Two independent experiments showed that combination of everolimus and PTK/ZK caused at least additive increases in anti-tumour activity compared to either monotherapy, without increases in toxicity. Pooling the data to improve the statistical power demonstrated the interactions to be synergistic. PK modelling showed that although PTK/ZK increased everolimus plasma concentrations by about twofold, this PK drug-drug interaction could not account for the increased anti-tumour effect of the combination. Modelling of the PTK/ZK dose-response curve in this model suggested that any effect of everolimus on the PK of PTK/ZK was unlikely to affect efficacy. Measurement of changes in tumour and plasma VEGF levels at the endpoint of therapy confirmed earlier observations of differential effects of these two agents., Conclusions: The combination of everolimus and PTK/ZK hold promise for the treatment of human cancers.

The aim was to determine the potential of the allosteric mammalian target of rapamycin inhibitor, everolimus, to act in combination with cytotoxic anticancer compounds in vitro and in vivo. A concomitant combination in vitro showed no evidence of antagonism, but enhanced the antiproliferative effects (additive to synergistic) with cisplatin, doxorubicin, 5-fluorouracil, gemcitabine, paclitaxel, and patupilone. Everolimus (1-5 mg/kg/d orally) was evaluated for antitumor activity in vivo alone or in combination with suboptimal cytotoxic doses using athymic nude mice bearing subcutaneous human H-596 lung, KB-31 cervical, or HCT-116 colon tumor xenografts. Everolimus monotherapy was very well tolerated and caused inhibition of tumor growth, rather than regression, and this was associated with a dose-dependent decline in tumor pS6 levels, a key downstream protein of mammalian target of rapamycin. At the doses used, the cytotoxics inhibited tumor growth and caused tolerable body-weight loss. Concomitant combinations of cisplatin, doxorubicin, paclitaxel, or patupilone with everolimus produced cooperative antitumor effects, in some cases producing regressions without clinically significant increases in toxicity. In contrast, combinations with gemcitabine and 5-fluorouracil were less well tolerated. Alternative administration schedules were tested for cisplatin, gemcitabine, or paclitaxel combined with everolimus: these did not dramatically affect cisplatin or gemcitabine activity or tolerability but were antagonistic for paclitaxel. Everolimus showed promising maintenance activity after treatment with doxorubicin or paclitaxel ceased. Overall, the results confirm that everolimus is an effective, well-tolerated suppressor of experimental human tumor growth, and although it did not show strong potentiation of efficacy, antitumor activity in vivo was increased without marked increases in toxicity, supporting clinical use of everolimus as a partner for conventional cytotoxics.

Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[(18)F]fluoro-glucose (FDG) and 3'-deoxy-3'-[(18)F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC(50) values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 microM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing.

Purpose: Comparative pharmacokinetic (PK) analysis of the mTOR inhibitor RAD001 (everolimus) in rats and mice., Methods: Blood cell partitioning, plasma protein binding and PK parameters of RAD001 in blood and tissues (including brain) of both mice and rats were determined. PK modeling predicted plasma/blood and tumor levels from a variety of regimens and these were compared with the known human PK profile. DCE-MRI was used to compare tumor vascularity between mice and rats. Estimation of IC50 values in vitro and ED50 values in vivo were used to provide an indication of anti-tumor activity., Results: The PK properties of RAD001 differed between mice and rats, including erythrocyte partitioning, plasma protein binding, plasma/blood t(1/2), oral bioavailability, volume of distribution, tissue/tumor penetration and elimination. Modeling of tumor and blood/plasma PK suggested that in mice, multiple daily administrations result in a 2-fold increase in tumor levels of RAD001 at steady state, whereas in rats, a 7.9-fold increase would occur. Weekly high-dose regimens were predicted not to facilitate tumor accumulation in either species. Total tumor levels of RAD001 were four- to eight-fold greater in rats than in mice. Rat tumors had a >2-fold greater plasma content and permeability compared to mouse tumors, which could contribute to differences in tumor drug uptake. Maximal antitumor effects (T/C of 0.04-0.35) were observed in both species after daily administration with similar C(max) and AUC values of unbound (free) RAD001. These free levels of RAD001 are exceeded in serum from cancer patients receiving clinically beneficial daily regimens. In rodents, brain penetration of RAD001 was poor, but was dose-dependent and showed over-proportional uptake in rats with a longer t(1/2) compared to the systemic circulation., Conclusions: The PK of RAD001 differed between mice and rats, with rats having a PK profile closer to that of humans. High intermittent doses of RAD001 may be more appropriate for treatment of brain tumors.

A central sensor of the availability of growth factors, nutrients and energy sources, the mammalian target of rapamycin complex 1 (mTORC1) kinase plays a key role in tumor biology. Consequently, mTORC1 inhibitors have been shown to have broad antitumor activity pre-clinically in experimental tumor models as well as clinically in cancer patients. Strikingly, certain tumor types appear to be predisposed to respond to mTORC1 inhibition, a phenomenon related to deregulation of critical elements of the PI3K/mTORC1 pathway. In this review we address optimization of clinical development in the context of mTORC1 inhibitor-induced activation of survival pathways, crosstalk between different signaling modules involved in malignant transformation, definition of rational target combination scenarios and biologically based dosing and patient stratification strategies. Emphasis is given where possible to mTORC1 drug development decisions based on full clinical publications.

Mammalian target of rapamycin (mTOR) regulates cellular processes important for progression of human cancer. RAD001 (everolimus), an mTORC1 (mTOR/raptor) inhibitor, has broad antitumor activity in preclinical models and cancer patients. Although most tumor lines are RAD001 sensitive, some are not. Selective mTORC1 inhibition can elicit increased AKT S473 phosphorylation, involving insulin receptor substrate 1, which is suggested to potentially attenuate effects on tumor cell proliferation and viability. Rictor may also play a role because rictor kinase complexes (including mTOR/rictor) regulate AKT S473 phosphorylation. The role of raptor and rictor in the in vitro response of human cancer cells to RAD001 was investigated. Using a large panel of cell lines representing different tumor histotypes, the basal phosphorylation of AKT S473 and some AKT substrates was found to correlate with the antiproliferative response to RAD001. In contrast, increased AKT S473 phosphorylation induced by RAD001 did not correlate. Similar increases in AKT phosphorylation occurred following raptor depletion using siRNA. Strikingly, rictor down-regulation attenuated AKT S473 phosphorylation induced by mTORC1 inhibition. Further analyses showed no relationship between modulation of AKT phosphorylation on S473 and T308 and AKT substrate phosphorylation patterns. Using a dual pan-class I phosphatidylinositol 3-kinase/mTOR catalytic inhibitor (NVP-BEZ235), currently in phase I trials, concomitant targeting of these kinases inhibited AKT S473 phosphorylation, eliciting more profound cellular responses than mTORC1 inhibition alone. However, reduced cell viability could not be predicted from biochemical or cellular responses to mTORC1 inhibitors. These data could have implications for the clinical application of phosphatidylinositol 3-kinase/mTOR inhibitors.