Semi-quantitative mass spectrometry in AML cells identifies new non-genomic targets of the EZH2 methyltransferase

Alterations to the gene encoding the EZH2 (KMT6A) methyltransferase, including both gain-of-function and loss-of-function, have been linked to a variety of haematological malignancies and solid tumours, suggesting a complex, context-dependent role of this methyltransferase. The successful implementation of molecularly targeted therapies against EZH2 requires a greater understanding of the potential mechanisms by which EZH2 contributes to cancer. One aspect of this effort is the mapping of EZH2 partner proteins and cellular targets. To this end we performed affinity-purification mass spectrometry in the FAB-M2 HL-60 acute myeloid leukaemia (AML) cell line before and after all-transretinoic acid-induced differentiation. These studies identified new EZH2 interaction partners and potential non-histone substrates for EZH2-mediated methylation. Our results suggest that EZH2 is involved in the regulation of translation through interactions with a number of RNA binding proteins and by methylating key components of protein synthesis such as eEF1A1. Given that deregulated mRNA translation is a frequent feature of cancer and that eEF1A1 is highly expressed in many human tumours, these findings present new possibilities for the therapeutic targeting of EZH2 in AML

Cohesin-independent STAG proteins interact with RNA and localise to R-loops to promote complex loading

Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA and are localised to R-loops where they contribute to R-loop regulation. Our results place SA1 within R-loop domains upstream of the cohesin complex and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease

Cohesin-independent STAG proteins interact with RNA and R-loops and promote complex loading

Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA, RNA binding proteins and R-loops, even in the absence of cohesin. Our results place SA1 on chromatin upstream of the cohesin ring and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease

Corporate Culture and Empathy and Excitement Labor of Service Employees inService Company, Mainly at Tokyo Disneyland

東京ディズニーランド(ディズニーシーを含むパーク)の大成功(集客と驚異的リピート率)の要因は、①「夢と魔法の王国」にふさわしいアトラクション1、②接客従業員(主に、非正社員、キャラクターを含む)のホスピタリティ・サービスが、顧客に「素晴らしい思い出に残る感動経験」を与えていることである。望ましいサービス労働のあり方は「顧客・従業員インターラクティブの共感に基づく従業員の感動労働」であるという仮説をたて、その解明を研究目的とした。①先行研究の考察、②運営会社へのインタビュー、③現場でのキャストのサービス労働の実査と簡単な質問、④顧客へのヒストリカル・インタビュー・アンケート実施という研究方法によって、接客従業員の「共感・感動労働」を実証中である。共感・感動労働の視点で、東京ディズニーランドと日本マクドナルド、スターバックスコーヒーとを比較した

Tumour kinome re-wiring governs resistance to palbociclib in oestrogen receptor positive breast cancers, highlighting new therapeutic modalities.

Combination of CDK4/6 inhibitors and endocrine therapy improves clinical outcome in advanced oestrogen receptor (ER)-positive breast cancer, however relapse is inevitable. Here, we show in model systems that other than loss of RB1 few gene-copy number (CN) alterations are associated with irreversible-resistance to endocrine therapy and subsequent secondary resistance to palbociclib. Resistance to palbociclib occurred as a result of tumour cell re-wiring leading to increased expression of EGFR, MAPK, CDK4, CDK2, CDK7, CCNE1 and CCNE2. Resistance altered the ER genome wide-binding pattern, leading to decreased expression of 'classical' oestrogen-regulated genes and was accompanied by reduced sensitivity to fulvestrant and tamoxifen. Persistent CDK4 blockade decreased phosphorylation of tuberous sclerosis complex 2 (TSC2) enhancing EGFR signalling, leading to the re-wiring of ER. Kinome-knockdown confirmed dependency on ERBB-signalling and G2/M-checkpoint proteins such as WEE1, together with the cell cycle master regulator, CDK7. Noteworthy, sensitivity to CDK7 inhibition was associated with loss of ER and RB1 CN. Overall, we show that resistance to CDK4/6 inhibitors is dependent on kinase re-wiring and the redeployment of signalling cascades previously associated with endocrine resistance and highlights new therapeutic networks that can be exploited upon relapse after CDK4/6 inhibition

A small-molecule PI3Kα activator for cardioprotection and neuroregeneration

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1,2,3,4,5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia–reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development

Discovery of naturally occurring ESR1 mutations in breast cancer cell lines modelling endocrine resistance.

Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance.Cancer Research U