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Abstract Background The product of the retinoblastoma-susceptibility gene (pRb) is a substrate for Protein Phosphatase 1 (PP1). At mitotic exit, all three PP1 isoforms, α, γ1 and δ, bind to pRb and dephosphorylate its Ser/Thr sites in a sequential and site-specific way. The pRb-C terminal has been reported to be necessary and sufficient for PP1α binding. The present study investigated whether the three PP1 isoforms from mitotic or asynchronous HeLa cells associate differentially with wild-type and pRb mutants, as well as the holoenzyme composition of the pRb-directed PP1. Results The requirement for the entire pRb molecule to achieve optimal PP1-binding was indicated by the fact that full-length pRb displayed the highest affinity for all three PP1 isoforms. Ser/Thr-to-Ala substitution for up to 14 pRb sites did not affect the ability of pRb to bind the PP1 isoforms derived from mitotic or asynchronous HeLa cells, thus suggesting that the phosphate-accepting residues on pRb do not regulate the interaction with PP1. To probe for the presence of PP1 targeting subunits in the pRb-directed PP1 complex, PP1 from mitotic or asynchronous HeLa cells was isolated by affinity chromatography on GST-Rb (either full-length or its deletion mutants Rb-big pocket or Rb-C-terminal). The PP1 was always obtained as free catalytic subunit, displaying all three isoforms, thus suggesting direct interaction between pRb and PP1. The direct association was confirmed by the ability of pRb to pull-down purified PP1 catalytic subunits and by in vitro reconstitution of a complex between PP1 catalytic subunit and the pRb-C-terminal. Conclusion The work indicated that the full length of the pRb molecule is required for optimal interaction with the PP1 isoforms and that the association between pRb and PP1 isoforms is direct.

Desmoplastic small round cell tumor (DSRCT) is an aggressive sarcoma subtype that is driven by the EWS-WT1 chimeric transcription factor. The prognosis for DSRCT is poor, and major advances in treating DSRCT have not occurred for over two decades. To identify effective therapeutic approaches to target DSRCT, we conducted a high-throughput drug sensitivity screen in a DSRCT cell line assessing chemosensitivity profiles for 79 small-molecule inhibitors. DSRCT cells were sensitive to PARP inhibitors (PARPi) and ataxia-telangiectasia and Rad3-related inhibitors (ATRi), as monotherapies and in combination. These effects were recapitulated using multiple clinical PARPi and ATRi in three biologically distinct, clinically relevant models of DSRCT, including cell lines, a patient-derived xenograft-derived organoid model, and a cell line-derived xenograft mouse model. Mechanistically, exposure to a combination of PARPi and ATRi caused increased DNA damage, G2-M checkpoint activation, micronuclei accumulation, replication stress, and R-loop formation. EWS-WT1 silencing abrogated these phenotypes and was epistatic with exogenous expression of the R-loop resolution enzyme RNase H1 in reversing sensitivity to PARPi and ATRi monotherapies. The combination of PARPi and ATRi also induced EWS-WT1-dependent cell-autonomous activation of the cyclic GMP-AMP synthase-stimulator of IFN genes innate immune pathway and cell-surface expression of PD-L1. Taken together, these findings point toward a role for EWS-WT1 in generating R-loop-dependent replication stress that leads to a targetable vulnerability, providing a rationale for the clinical assessment of PARPi and ATRi in DSRCT. Significance: EWS-WT1, the unique oncogenic driver of desmoplastic small round cell tumors, confers sensitivity to PARP and ATR inhibitors, supporting the potential of these drugs in treating patients with this aggressive sarcoma subtype., (©2024 The Authors; Published by the American Association for Cancer Research.)

The role of endothelial cells in promoting cancer cell extravasation to the brain during the interaction of cancer cells with the vasculature is not well characterised. We show that brain endothelial cells activate EGFR signalling in triple-negative breast cancer cells with propensity to metastasise to the brain. This activation is dependent on soluble factors secreted by brain endothelial cells, and occurs via the RAC1 GEF DOCK4, which is required for breast cancer cell extravasation to the brain in vivo. Knockdown of DOCK4 inhibits breast cancer cell entrance to the brain without affecting cancer cell survival or growth. Defective extravasation is associated with loss of elongated morphology preceding intercalation into brain endothelium. We also show that brain endothelial cells promote paracrine stimulation of mesenchymal-like morphology of breast cancer cells via DOCK4, DOCK9, RAC1 and CDC42. This stimulation is accompanied by EGFR activation necessary for brain metastatic breast cancer cell elongation which can be reversed by the EGFR inhibitor Afatinib. Our findings suggest that brain endothelial cells promote metastasis through activation of cell signalling that renders breast cancer cells competent for extravasation. This represents a paradigm of brain endothelial cells influencing the signalling and metastatic competency of breast cancer cells., (© 2024. The Author(s).)

Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have revolutionized the treatment of hormone-positive metastatic breast cancers (mBCs). They are currently established as standard therapies in combination with endocrine therapy as first- and second-line systemic treatment options for both endocrine-sensitive and endocrine-resistant mBC populations. In the first-line metastatic setting, the median progression-free survival for the three currently approved CDK4/6 inhibitors, palbociclib, ribociclib, and abemaciclib, with aromatase inhibitors is greater than 2 years (palbociclib 27.6 months; ribociclib 25.3 months; and abemaciclib 28.18 months). Although CDK4/6 inhibitors have significant clinical benefits and enable physicians to delay starting chemotherapy, they are expensive and can be associated with drug toxicities. Here, we have performed a systemic review of the reported molecular markers predictive of drug response including intrinsic and acquired resistance for CDK4/6 inhibition in mBC. The rapidly emerging molecular landscape is captured through next-generation sequencing of breast cancers (DNA with or without RNA), liquid biopsies (circulating tumor DNA), and protein analyses. Individual molecular candidates with robust and reliable evidence are discussed in more depth., Competing Interests: Rebecca RoylanceLeadership: Google healthHonoraria: Daiichi Sankyo Europe GmbH, Daiichi Sankyo/Astra Zeneca, Daiichi Sankyo/Astra ZenecaConsulting or Advisory Role: Lilly, Daiichi Sankyo Europe GmbH, IQVIA, Daiichi Sankyo/Astra ZenecaTravel, Accommodations, Expenses: Roche/Genentech, Daiichi Sankyo Europe GmbH, Daiichi Sankyo Europe GmbH, BMSNo other potential conflicts of interest were reported.

Loss-of-function mutations in the RB1 tumour suppressor are key drivers in cancer, including osteosarcoma. RB1 loss-of-function compromises genome-maintenance and hence could yield vulnerability to therapeutics targeting such processes. Here we demonstrate selective hypersensitivity to clinically-approved inhibitors of Poly-ADP-Polymerase1,2 inhibitors (PARPi) in RB1-defective cancer cells, including an extended panel of osteosarcoma-derived lines. PARPi treatment results in extensive cell death in RB1-defective backgrounds and prolongs survival of mice carrying human RB1-defective osteosarcoma grafts. PARPi sensitivity is not associated with canonical homologous recombination defect (HRd) signatures that predict PARPi sensitivity in cancers with BRCA1,2 loss, but is accompanied by rapid activation of DNA replication checkpoint signalling, and active DNA replication is a prerequisite for sensitivity. Importantly, sensitivity in backgrounds with natural or engineered RB1 loss surpasses that seen in BRCA-mutated backgrounds where PARPi have established clinical benefit. Our work provides evidence that PARPi sensitivity extends beyond cancers identifiable by HRd and advocates PARP1,2 inhibition as a personalised strategy for RB1-mutated osteosarcoma and other cancers., (© 2021. The Author(s).)

Cellular senescence is a stable cell cycle arrest that normal cells undergo after a finite number of divisions, in response to a variety of intrinsic and extrinsic stimuli. Although senescence is largely established and maintained by the p53/p21 WAF1/CIP1 and pRB/p16 INK4A tumour suppressor pathways, the downstream targets responsible for the stability of the growth arrest are not known. We have employed a stable senescence bypass assay in conditionally immortalised human breast fibroblasts (CL3 EcoR ) to investigate the role of the DREAM complex and its associated components in senescence. DREAM is a multi-subunit complex comprised of the MuvB core, containing LIN9, LIN37, LIN52, LIN54, and RBBP4, that when bound to p130, an RB1 like protein, and E2F4 inhibits cell cycle-dependent gene expression thereby arresting cell division. Phosphorylation of LIN52 at Serine 28 is required for DREAM assembly. Re-entry into the cell cycle upon phosphorylation of p130 leads to disruption of the DREAM complex and the MuvB core, associating initially to B-MYB and later to FOXM1 to form MMB and MMB-FOXM1 complexes respectively. Here we report that simultaneous expression of MMB-FOXM1 complex components efficiently bypasses senescence with LIN52, B-MYB, and FOXM1 as the crucial components. Moreover, bypass of senescence requires non-phosphorylated LIN52 that disrupts the DREAM complex, thereby indicating a central role for assembly of the DREAM complex in senescence., (© 2021. The Author(s).)

To avoid replicative senescence or telomere-induced apoptosis, cancers employ telomere maintenance mechanisms (TMMs) involving either the upregulation of telomerase or the acquisition of recombination-based alternative telomere lengthening (ALT). The choice of TMM may differentially influence cancer evolution and be exploitable in targeted therapies. Here, we examine TMMs in a panel of 17 osteosarcoma-derived cell lines, defining three separate groups according to TMM and the length of telomeres maintained. Eight were ALT-positive, including the previously uncharacterized lines, KPD and LM7. While ALT-positive lines all showed excessive telomere length, ALT-negative cell lines fell into two groups according to their telomere length: HOS-MNNG, OHSN, SJSA-1, HAL, 143b, and HOS displayed subnormally short telomere length, while MG-63, MHM, and HuO-3N1 displayed long telomeres. Hence, we further subcategorized ALT-negative TMM into long-telomere (LT) and short-telomere (ST) maintenance groups. Importantly, subnormally short telomeres were significantly associated with hypersensitivity to three different therapeutics targeting the protein kinase ataxia telangiectasia and Rad3-related (ATR) (AZD-6738/Ceralasertib, VE-822/Berzoserib, and BAY-1895344) compared to long telomeres maintained via ALT or telomerase. Within 24 h of ATR inhibition, cells with short but not long telomeres displayed chromosome bridges and underwent cell death, indicating a selective dependency on ATR for chromosome stability. Collectively, our work provides a resource to identify links between the mode of telomere maintenance and drug sensitivity in osteosarcoma and indicates that telomere length predicts ATR inhibitor sensitivity in cancer., Competing Interests: The authors declare no competing financial interest., (© 2020 American Chemical Society.)

Deregulation of cyclin-dependent kinases 4 and 6 (CDK4/6) is highly prevalent in cancer; yet, inhibitors against these kinases are currently used only in restricted tumour contexts. The extent to which cancers depend on CDK4/6 and the mechanisms that may undermine such dependency are poorly understood. Here, we report that signalling engaging the MET proto-oncogene receptor tyrosine kinase/focal adhesion kinase (FAK) axis leads to CDK4/6-independent CDK2 activation, involving as critical mechanistic events loss of the CDKI p21 CIP1 and gain of its regulator, the ubiquitin ligase subunit SKP2. Combined inhibition of MET/FAK and CDK4/6 eliminates the proliferation capacity of cancer cells in culture, and enhances tumour growth inhibition in vivo. Activation of the MET/FAK axis is known to arise through cancer extrinsic and intrinsic cues. Our work predicts that such cues support cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases and identifies MET/FAK as a tractable route to broaden the utility of CDK4/6 inhibitor-based therapies in the clinic.

High-content imaging connects the information-rich method of microscopy with the systematic objective principles of software-driven analysis. Suited to automation and, therefore, considerable scale-up of study size, this approach can deliver multiparametric data over cell populations or at the level of the individual cell and has found considerable utility in reverse genetic and pharmacological screens. Here we present a method to screen small interfering RNA (siRNA) libraries allowing subsequent observation of the impact of each knockdown on two interlinked, high-content, G1-/S-phase cell cycle transition assays related to cyclin-dependent kinase (CDK) 2 activity. We show how plasticity within the network governing the activity of this kinase can be detected by combining modifier siRNAs with a siRNA library. The method uses fluorescent immunostaining of a nuclear antigen, CyclinA, following cell fixation while also preserving the fluorescence of a stably expressed fluorescent protein-tagged reporter for CDK2 activity. We provide methodology for data extraction and handling including an R-script that converts the multidimensional data into four simple binary outcomes, on which a hit-mining strategy can be built. The workflow described can in principle be adopted to yield quantitative single-cell-resolved data and mining for outcomes relating to a broad range of other similar readouts and signaling contexts.

Glycine transporter 1 (GlyT1) plays a crucial role in regulating extracellular glycine concentrations and might thereby constitute a new drug target for the modulation of glycinergic inhibition in pain signaling. Consistent with this view, inhibition of GlyT1 has been found to induce antinociceptive effects in various animal pain models. We have shown previously that the lidocaine metabolite N-ethylglycine (EG) reduces GlyT1-dependent glycine uptake by functioning as an artificial substrate for this transporter. Here, we show that EG is specific for GlyT1 and that in rodent models of inflammatory and neuropathic pain, systemic treatment with EG results in an efficient amelioration of hyperalgesia and allodynia without affecting acute pain. There was no effect on motor coordination or the development of inflammatory edema. No adverse neurological effects were observed after repeated high-dose application of EG. EG concentrations both in blood and spinal fluid correlated with an increase of glycine concentration in spinal fluid. The time courses of the EG and glycine concentrations corresponded well with the antinociceptive effect. Additionally, we found that EG reduced the increase in neuronal firing of wide-dynamic-range neurons caused by inflammatory pain induction. These findings suggest that systemically applied lidocaine exerts antihyperalgesic effects through its metabolite EG in vivo, by enhancing spinal inhibition of pain processing through GlyT1 modulation and subsequent increase of glycine concentrations at glycinergic inhibitory synapses. EG and other substrates of GlyT1, therefore, may be a useful therapeutic agent in chronic pain states involving spinal disinhibition.

Loss of retinoblastoma protein (RB1) function is a major driver in cancer development. We have recently reported that, in addition to its well-documented functions in cell cycle and fate control, RB1 and its paralogs have a novel role in regulating DNA repair by non-homologous end joining (NHEJ). Here we summarize our findings and present mechanistic hypotheses on how RB1 may support the DNA repair process and the therapeutic implications for patients who harbor RB1-negative cancers.

Deficiencies in DNA double-strand break (DSB) repair lead to genetic instability, a recognized cause of cancer initiation and evolution. We report that the retinoblastoma tumor suppressor protein (RB1) is required for DNA DSB repair by canonical non-homologous end-joining (cNHEJ). Support of cNHEJ involves a mechanism independent of RB1's cell-cycle function and depends on its amino terminal domain with which it binds to NHEJ components XRCC5 and XRCC6. Cells with engineered loss of RB family function as well as cancer-derived cells with mutational RB1 loss show substantially reduced levels of cNHEJ. RB1 variants disabled for the interaction with XRCC5 and XRCC6, including a cancer-associated variant, are unable to support cNHEJ despite being able to confer cell-cycle control. Our data identify RB1 loss as a candidate driver of structural genomic instability and a causative factor for cancer somatic heterogeneity and evolution., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators. Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software(1) to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

The retinoblastoma susceptibility protein RB1 is a key regulator of cell proliferation and fate. RB1 operates through nucleating the formation of multi-component protein complexes involved in the regulation of gene transcription, chromatin structure and protein stability. Phosphorylation of RB1 by cyclin-dependent kinases leads to conformational alterations and inactivates the capability of RB1 to bind partner protein. Using small angle X-ray scattering in combination with single particle analysis of transmission electron microscope images of negative-stained material we present the first three-dimensional reconstruction of non-phosphorylated RB1 revealing an extended architecture and deduce the domain arrangement within the molecule. Phosphorylation results in an overt alteration of the molecular shape and dimensions, consistent with the transition to a compact globular architecture. The work presented provides what is to our knowledge the first description of the relative domain arrangement in active RB1 and predicts the molecular movement that leads to RB1 inactivation following protein phosphorylation.