Temporal regulation of microtubule dynamics is essential for proper progression of mitosis and control of microtubule plus-end tracking proteins by phosphorylation is an essential component of this regulation. Here we show that Aurora B and CDK1 phosphorylate microtubule end-binding protein 2 (EB2) at multiple sites within the amino terminus and a cluster of serine/threonine residues in the linker connecting the calponin homology and end-binding homology domains. EB2 phosphorylation, which is strictly associated with mitotic entry and progression, reduces the binding affinity of EB2 for microtubules. Expression of non-phosphorylatable EB2 induces stable kinetochore microtubule dynamics and delays formation of bipolar metaphase plates in a microtubule binding-dependent manner, and leads to aneuploidy even in unperturbed mitosis. We propose that Aurora B and CDK1 temporally regulate the binding affinity of EB2 for microtubules, thereby ensuring kinetochore microtubule dynamics, proper mitotic progression and genome stability.

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The high specificity of antibodies for their antigen allows a fine discrimination of target conformations and post-translational modifications, making antibodies the first choice tool to interrogate the proteome. We describe here an approach based on a large-scale intracellular expression and selection of antibody fragments in eukaryotic cells, so-called intrabodies, and the subsequent identification of their natural target within living cell. Starting from a phenotypic trait, this integrated system allows the identification of new therapeutic targets together with their companion inhibitory intrabody. We applied this system in a model of allergy and inflammation. We first cloned a large and highly diverse intrabody library both in a plasmid and a retroviral eukaryotic expression vector. After transfection in the RBL-2H3 rat basophilic leukemia cell line, we performed seven rounds of selection to isolate cells displaying a defect in FcεRI-induced degranulation. We used high throughput sequencing to identify intrabody sequences enriched during the course of selection. Only one intrabody was common to both plasmid and retroviral selections, and was used to capture and identify its target from cell extracts. Mass spectrometry analysis identified protein RGD1311164 (C12orf4), with no previously described function. Our data demonstrate that RGD1311164 is a cytoplasmic protein implicated in the early signaling events following FcεRI-induced cell activation. This work illustrates the strength of the intrabody-based in-cell selection, which allowed the identification of a new player in mast cell activation together with its specific inhibitor intrabody.

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A mathematical model capable of accurately characterizing intracellular disposition of ADCs is essential for a priori predicting unconjugated drug concentrations inside the tumor. Towards this goal, the objectives of this manuscript were to: (1) evolve previously published cellular disposition model of ADC with more intracellular details to characterize the disposition of T-DM1 in different HER2 expressing cell lines, (2) integrate the improved cellular model with the ADC tumor disposition model to a priori predict DM1 concentrations in a preclinical tumor model, and (3) identify prominent pathways and sensitive parameters associated with intracellular activation of ADCs. The cellular disposition model was augmented by incorporating intracellular ADC degradation and passive diffusion of unconjugated drug across tumor cells. Different biomeasures and chemomeasures for T-DM1, quantified in the companion manuscript, were incorporated into the modified model of ADC to characterize in vitro pharmacokinetics of T-DM1 in three HER2+ cell lines. When the cellular model was integrated with the tumor disposition model, the model was able to a priori predict tumor DM1 concentrations in xenograft mice. Pathway analysis suggested different contribution of antigen-mediated and passive diffusion pathways for intracellular unconjugated drug exposure between in vitro and in vivo systems. Global and local sensitivity analyses revealed that non-specific deconjugation and passive diffusion of the drug across tumor cell membrane are key parameters for drug exposure inside a cell. Finally, a systems pharmacokinetic model for intracellular processing of ADCs has been proposed to highlight our current understanding about the determinants of ADC activation inside a cell.

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An analysis of oncology regulatory approvals since 2011 reveals that several new drugs demonstrate exceptional clinical activity in Phase 1 or Phase 2 trials. This has led to increased use of regulatory mechanisms that allow initial approval based on these early data. However, full regulatory approval is contingent upon subsequent verification of clinical benefit in confirmatory Phase 3 randomized controlled trials.There has been a growing interest in the systematic collection of real-world data to gain further insight into the impact of new drugs as they are introduced into clinical practice. Furthermore, payers, physicians, and patients increasingly require more data on alternative comparators, specific subgroups, patient-reported outcomes, and long-term toxicities – data that may be more appropriately collected in real-world studies than in separate confirmatory trials.We propose that for oncology drugs that demonstrate "exceptional activity" in early trials and receive accelerated/conditional approval and/or Breakthrough Therapy Designation, and for certain expanded indications, regulatory authorities should consider accepting data from prospectivelyagreed pragmatic randomized clinical trials (pRCTs) to grant full regulatory approval. The "exceptional activity" would be defined as (1) an objective response rate ≥50% to a single agent or (2) a hazard ratio of <0.5 in an early randomized study. In pRCTs, patients would be randomized to two or more interventions and then treated and followed up according to the investigators’ usual practice. Patients participating in pRCTs would be representative of the real-world population. pRCTs would provide data necessary for full regulatory approval and would address questions of the new drug’s value for reimbursement purposes.
© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].

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In the PALOMA-3 study, palbociclib plus fulvestrant demonstrated improved progression-free survival compared with fulvestrant plus placebo in HR+/HER2- endocrine-resistant metastatic breast cancer (MBC). This analysis compared patient reported outcomes (PROs) between the two treatment groups.
Patients were randomized 2:1 to receive palbociclib 125 mg/day orally for 3 weeks followed by 1 week off (n=347) plus fulvestrant (500 mg intramuscularly per standard of care) or placebo plus fulvestrant (n=174). PROs were assessed on Day 1 of Cycles 1-4 and of every other subsequent cycle starting with Cycle 6 using the EORTC QLQ-C30 and its breast cancer module, QLQ-BR23. High scores (range 0-100) could indicate better functioning/quality of life (QoL) or worse symptom severity. Repeated measures mixed-effects analyses were performed to compare on-treatment overall scores and changes from baseline between treatment groups while controlling for baseline. Between-group comparisons of time to deterioration in global QoL and pain were made using an unstratified log-rank test and Cox proportional hazards model.
Questionnaire completion rates were high at baseline and during treatment (from baseline to Cycle 14, ≥95.8% in each group completed ≥1 question on the EORTC QLQ-C30). On treatment, estimated overall global QoL scores significantly favored the palbociclib plus fulvestrant group (66.1, 95% confidence interval [CI]: 64.5, 67.7 vs 63.0, 95% CI: 60.6, 65.3; P=0.0313). Significantly greater improvement from baseline in pain was also observed in this group (-3.3, 95% CI: -5.1,-1.5 vs 2.0, 95% CI:-0.6, 4.6; P=0.0011). No significant differences were observed for other QLQ-BR 23 functioning domains, breast, or arm symptoms. Treatment with palbociclib plus fulvestrant significantly delayed deterioration in global QoL (P<0.025) and pain (P <0.001) compared with fulvestrant alone.
Palbociclib plus fulvestrant allowed patients to maintain good QoL in the endocrine resistance setting while experiencing substantially delayed disease progression.
ClinicalTrials.gov identifier NCT01942135.
© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

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