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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.

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 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.

Background Therapies targeting estrogenic stimulation in estrogen receptor-positive (ER+) breast cancer (BC) reduce mortality, but resistance remains a major clinical problem. Molecular studies have shown few high-frequency mutations to be associated with endocrine resistance. In contrast, expression profiling of primary ER+ BC samples has identified several promising signatures/networks for targeting. Methods To identify common adaptive mechanisms associated with resistance to aromatase inhibitors (AIs), we assessed changes in global gene expression during adaptation to long-term estrogen deprivation (LTED) in a panel of ER+ BC cell lines cultured in 2D on plastic (MCF7, T47D, HCC1428, SUM44 and ZR75.1) or in 3D on collagen (MCF7) to model the stromal compartment. Furthermore, dimethyl labelling followed by LC-MS/MS was used to assess global changes in protein abundance. The role of target genes/proteins on proliferation, ER-mediated transcription and recruitment of ER to target gene promoters was analysed. Results The cholesterol biosynthesis pathway was the common upregulated pathway in the ER+ LTED but not the ER– LTED cell lines, suggesting a potential mechanism dependent on continued ER expression. Targeting the individual genes of the cholesterol biosynthesis pathway with siRNAs caused a 30–50 % drop in proliferation. Further analysis showed increased expression of 25-hydroxycholesterol (HC) in the MCF7 LTED cells. Exogenous 25-HC or 27-HC increased ER-mediated transcription and expression of the endogenous estrogen-regulated gene TFF1 in ER+ LTED cells but not in the ER– LTED cells. Additionally, recruitment of the ER and CREB-binding protein (CBP) to the TFF1 and GREB1 promoters was increased upon treatment with 25-HC and 27-HC. In-silico analysis of two independent studies of primary ER+ BC patients treated with neoadjuvant AIs showed that increased expression of MSMO1 , EBP , LBR and SQLE enzymes, required for cholesterol synthesis and increased in our in-vitro models, was significantly associated with poor response to endocrine therapy. Conclusion Taken together, these data provide support for the role of cholesterol biosynthesis enzymes and the cholesterol metabolites, 25-HC and 27-HC, in a novel mechanism of resistance to endocrine therapy in ER+ BC that has potential as a therapeutic target.

Antibody combinations targeting cell surface receptors are a new modality of cancer therapy. The trafficking and signalling mechanisms regulated by such therapeutics are not fully understood but could underlie differential tumour responses. We explored EGFR trafficking upon treatment with the antibody combination Sym004 which has shown promise clinically. Sym004 promoted EGFR endocytosis distinctly from EGF: it was asynchronous, not accompanied by canonical signalling events and involved EGFR clustering within detergent-insoluble plasma mebrane-associated tubules. Sym004 induced lysosomal degradation independently of EGFR ubiquitylation but dependent upon Hrs/Tsg101 that are required for the formation of intraluminal vesicles (ILVs) within late endosomes. We propose Sym004 cross-links EGFR physically triggering EGFR endocytosis and incorporation onto ILVs and so Sym004 sensitivity correlates with EGFR numbers available for binding, rather than specific signalling events. Consistently Sym004 efficacy and potentiation of cisplatin responses correlated with EGFR surface expression in head and neck cancer cells. These findings will have implications in understanding the mode of action of this new class of cancer therapeutics.

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. ESR1 mutations occur in endocrine-resistant patients but have not yet been reported in in vitro models of breast cancer. Here, the authors report the discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in two breast cancer cell lines after acquisition of resistance to long-term-estrogen-deprivation.

Primary cilia are antenna-like organelles which sense extracellular cues and act as signalling hubs. Cilia dysfunction causes a heterogeneous group of disorders known as ciliopathy syndromes affecting most organs. Cilia disassembly, the process by which cells lose their cilium, is poorly understood but frequently observed in disease and upon cell transformation. Here, we uncover a role for the PI3Kα signalling enzyme in cilia disassembly. Genetic PI3Kα-hyperactivation, as observed in PIK3CA -related overgrowth spectrum (PROS) and cancer, induced a ciliopathy-like phenotype during mouse development. Mechanistically, PI3Kα and PI3Kβ produce the PIP 3 lipid at the cilia transition zone upon disassembly stimulation. PI3Kα activation initiates cilia disassembly through a kinase signalling axis via the PDK1/PKCι kinases, the CEP170 centrosomal protein and the KIF2A microtubule-depolymerising kinesin. Our data suggest diseases caused by PI3Kα-activation may be considered ‘Disorders with Ciliary Contributions’, a recently-defined subset of ciliopathies in which some, but not all, of the clinical manifestations result from cilia dysfunction. Primary cilia are cell organelles disrupted in developmental diseases and cancer. Here, the authors identify cilia regulators that generate a specific cilia membrane lipid, which may help to better understand diseases caused by mutations in PI3-kinase subunits.

Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved “S259” inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.

Understanding cellular diversity and disease mechanisms requires a global analysis of proteins and their modifications. While next-generation sequencing has advanced our understanding of cellular heterogeneity, it fails to capture downstream signalling networks. Ultrasensitive mass spectrometry-based proteomics enables unbiased protein-level analysis of low cell numbers, down to single cells. However, phosphoproteomics remains limited to high-input samples due to sample losses and poor reaction efficiencies associated with processing low cell numbers. Isobaric stable isotope labelling is a promising approach for reproducible and accurate quantification of low abundant phosphopeptides. Here, we introduce SPARCE ( S treamlined P hosphoproteomic A nalysis of R are CE lls) for multiplexed phosphoproteomic analysis of low cell numbers. SPARCE integrates cell isolation, water-based lysis, on-tip TMT labelling, and phosphopeptide enrichment. SPARCE outperforms traditional methods by enhancing labelling efficiency and phosphoproteome coverage. To demonstrate the utility of SPARCE, we analysed four patient-derived glioblastoma stem cell lines, reliably quantifying phosphosite changes from 1000 FACS-sorted cells. This workflow expands the possibilities for signalling analysis of rare cell populations. Streamlined Phosphoproteomic Analysis of Rare CElls (SPARCE) enables robust quantification of proteomes and phosphoproteomes from just 1,000 FACS-sorted cells, advancing signalling analysis in rare populations.

The linear-ubiquitin chain assembly complex (LUBAC) modulates signalling via various immune receptors. In tumour necrosis factor (TNF) signalling, linear (also known as M1) ubiquitin enables full gene activation and prevents cell death. However, the mechanisms underlying cell death prevention remain ill-defined. Here, we show that LUBAC activity enables TBK1 and IKKε recruitment to and activation at the TNF receptor 1 signalling complex (TNFR1-SC). While exerting only limited effects on TNF-induced gene activation, TBK1 and IKKε are essential to prevent TNF-induced cell death. Mechanistically, TBK1 and IKKε phosphorylate the kinase RIPK1 in the TNFR1-SC, thereby preventing RIPK1-dependent cell death. This activity is essential in vivo, as it prevents TNF-induced lethal shock. Strikingly, NEMO (also known as IKKγ), which mostly, but not exclusively, binds the TNFR1-SC via M1 ubiquitin, mediates the recruitment of the adaptors TANK and NAP1 (also known as AZI2). TANK is constitutively associated with both TBK1 and IKKε, while NAP1 is associated with TBK1. We discovered a previously unrecognized cell death checkpoint that is mediated by TBK1 and IKKε, and uncovered an essential survival function for NEMO, whereby it enables the recruitment and activation of these non-canonical IKKs to prevent TNF-induced cell death. Lafont et al. uncover a checkpoint mediated by TBK1 and IKKε, which phosphorylate RIPK1 in the TNFR1-SC. TBK1 and IKKε recruitment depends on M1 ubiquitylation and NEMO to restrict TNF-induced cell death.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development 1 – 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. A new specific, small-molecule activator of the PI3Kα isoform (UCL-TRO-1938) identified through high-throughput screening can transiently activate PI3K signalling and biological responses in cells and tissues, with potential therapeutic applications in tissue protection and regeneration.