PAR Proteins Research Articles

Molecular dissection of the chromosome partitioning protein RocS and regulation by phosphorylation.

Chromosome segregation in bacteria is a critical process ensuring that each daughter cell receives an accurate copy of the genetic material during cell division. Active segregation factors, such as the ParABS system or SMC complexes, are usually essential for this process, but they are surprisingly dispensable in Streptococcus pneumoniae. Rather, chromosome segregation in S. pneumoniae relies on the protein Regulator of Chromosome Segregation (RocS), although the molecular mechanisms involved remain elusive. By combining genetics, in vivo imaging, and biochemical approaches, we dissected the molecular features of RocS involved in chromosome segregation. We investigated the respective functions of the three RocS domains, specifically the C-terminal amphipathic helix (AH), the N-terminal DNA-binding domain (DBD), and the coiled-coil domain (CCD) separating the AH and the DBD. Notably, we found that a single AH is not sufficient for membrane binding and that RocS requires prior oligomerization to interact with the membrane. We further demonstrated that this self-interaction was driven by the N-terminal part of the CCD. On the other hand, we revealed that the C-terminal part of the CCD corresponds to a domain of unknown function (DUF 536) and is defined by three conserved glutamines, which play a crucial role in RocS-mediated chromosome segregation. Finally, we showed that the DBD is phosphorylated by the unique serine-threonine kinase of S. pneumoniae StkP and that mimicking this phosphorylation abrogated RocS binding to DNA. Overall, this study offers new insights into chromosome segregation in Streptococci and paves the way for a deeper understanding of RocS-like proteins in other bacteria.IMPORTANCEBacteria have evolved a variety of mechanisms to properly segregate their genetic material during cell division. In this study, we performed a molecular dissection of the chromosome partitioning protein Regulator of Chromosome Segregation (RocS), a pillar element of chromosome segregation in S. pneumoniae that is also generally conserved in the Streptococcaceae family. Our systematic investigation sheds light on the molecular features required for successful pneumococcal chromosome segregation and the regulation of RocS by phosphorylation. In addition, our study also revealed that RocS shares functional domains with the Par protein, involved in an atypical plasmid segregation system. Therefore, we expect that our findings may serve to extend our understanding of RocS and RocS-like proteins while broadening the repertoire of partitioning systems used in bacteria.

Abstract PR-19: Cell polarity proteins as novel regulators of macropinocytosis

Abstract Macropinocytosis has emerged as a nutrient-scavenging pathway that cancer cells exploit to survive the nutrient-deprived conditions of the tumor microenvironment. Cancer cells are especially reliant on glutamine for their survival, and in pancreatic ductal adenocarcinoma (PDAC) cells, glutamine deficiency can enhance the stimulation of macropinocytosis, allowing the cells to escape metabolic stress through the production of extracellular-protein-derived amino acids. Here, we identify the atypical protein kinase C (aPKC) enzymes, PKCz and PKCi, as novel regulators of macropinocytosis. In normal epithelial cells, aPKCs are known to regulate cell polarity in association with the scaffold proteins Par3 and Par6, controlling the function of several targets, including the microtubule-associated Par1 kinases. In PDAC cells, we identify that each of these cell polarity proteins are required for glutamine stress-induced macropinocytosis. Mechanistically, we find that the aPKCs are regulated by EGFR signaling or by the transcription factor CREM to promote the relocation of Par3 to microtubules, facilitating macropinocytosis in a dynein-dependent manner. Importantly, we determine that cell fitness impairment caused by aPKC depletion in glutamine stress is rescued by the restoration of macropinocytosis and that aPKCs support PDAC growth in vivo. These results identify a previously unappreciated role for the cell polarity protein network in the regulation of macropinocytosis and provide a better understanding of the mechanistic underpinnings that control macropinocytic uptake in the context of metabolic stress. Citation Format: Guillem Lambies Barjau, Szu-Wei Lee, Karen Duong-Polk Duong-Polk, David Dawson, Cheska Marie Galapate, Swetha Maganti, Cosimo Commisso. Cell polarity proteins as novel regulators of macropinocytosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-19.

MInsc coordinates Par3 and NuMA condensates for assembly of the spindle orientation machinery in asymmetric cell division

As a fundamental process governing the self-renewal and differentiation of stem cells, asymmetric cell division is controlled by several conserved regulators, including the polarity protein Par3 and the microtubule-associated protein NuMA, which orchestrate the assembly and interplay of the Par3/Par6/mInsc/LGN complex at the apical cortex and the LGN/Gαi/NuMA/Dynein complex at the mitotic spindle to ensure asymmetric segregation of cell fate determinants. However, this model, which is well-supported by genetic studies, has been challenged by evidence of competitive interaction between NuMA and mInsc for LGN. Here, the solved crystal structure of the Par3/mInsc complex reveals that mInsc competes with Par6β for Par3, raising questions about how proteins assemble overlapping targets into functional macromolecular complexes. Unanticipatedly, we discover that Par3 can recruit both Par6β and mInsc by forming a dynamic condensate through phase separation. Similarly, the phase-separated NuMA condensate enables the coexistence of competitive NuMA and mInsc with LGN in the same compartment. Bridge by mInsc, Par3/Par6β and LGN/NuMA condensates coacervate, robustly enriching all five proteins both in vitro and within cells. These findings highlight the pivotal role of protein condensates in assembling multi-component signalosomes that incorporate competitive protein-protein interaction pairs, effectively overcoming stoichiometric constraints encountered in conventional protein complexes.

Oligomerization and positive feedback on membrane recruitment encode dynamically stable PAR-3 asymmetries in the C. elegans zygote.

Studies of PAR polarity have emphasized a paradigm in which mutually antagonistic PAR proteins form complementary polar domains in response to transient cues. A growing body of work suggests that the oligomeric scaffold PAR-3 can form unipolar asymmetries without mutual antagonism, but how it does so is largely unknown. Here we combine single molecule analysis and modeling to show how the interplay of two positive feedback loops promote dynamically stable unipolar PAR-3 asymmetries in early C. elegans embryos. First, the intrinsic dynamics of PAR-3 membrane binding and oligomerization encode negative feedback on PAR-3 dissociation. Second, membrane-bound PAR-3 promotes its own recruitment through a mechanism that requires the anterior polarity proteins CDC-42, PAR-6 and PKC-3. Using a kinetic model tightly constrained by our experimental measurements, we show that these two feedback loops are individually required and jointly sufficient to encode dynamically stable and locally inducible unipolar PAR-3 asymmetries in the absence of posterior inhibition. Given the central role of PAR-3, and the conservation of PAR-3 membrane-binding, oligomerization, and core interactions with PAR-6/aPKC, these results have widespread implications for PAR-mediated polarity in metazoa.

Loss of the polarity protein Par3 promotes dendritic spine neoteny and enhances learning and memory

The Par3 polarity protein is critical for subcellular compartmentalization in different developmental processes. Variants of PARD3, encoding PAR3, are associated with intelligence and neurodevelopmental disorders. However, the role of Par3 in glutamatergic synapse formation and cognitive functions invivo remains unknown. Here, we show that forebrain-specific Par3 conditional knockout leads to increased long, thin dendritic spines invivo. In addition, we observed a decrease in the amplitude of miniature excitatory postsynaptic currents. Surprisingly, loss of Par3 enhances hippocampal-dependent spatial learning and memory and repetitive behavior. Phosphoproteomic analysis revealed proteins regulating cytoskeletal dynamics are significantly dysregulated downstream of Par3. Mechanistically, we found Par3 deletion causes increased Rac1 activation and dysregulated microtubule dynamics through CAMSAP2. Together, our data reveal an unexpected role for Par3 as a molecular gatekeeper in regulating the pool of immature dendritic spines, a rate-limiting step of learning and memory, through modulating Rac1 activation and microtubule dynamics invivo.

A CDK1 phosphorylation site on Drosophila PAR-3 regulates neuroblast polarisation and sensory organ formation.

The generation of distinct cell fates during development depends on asymmetric cell division of progenitor cells. In the central and peripheral nervous system of Drosophila, progenitor cells respectively called neuroblasts or sensory organ precursors use PAR polarity during mitosis to control cell fate determination in their daughter cells. How polarity and the cell cycle are coupled, and how the cell cycle machinery regulates PAR protein function and cell fate determination is poorly understood. Here, we generate an analog sensitive allele of CDK1 and reveal that its partial inhibition weakens but does not abolish apical polarity in embryonic and larval neuroblasts and leads to defects in polarisation of fate determinants. We describe a novel in vivo phosphorylation of Bazooka, the Drosophila homolog of PAR-3, on Serine180, a consensus CDK phosphorylation site. In some tissular contexts, phosphorylation of Serine180 occurs in asymmetrically dividing cells but not in their symmetrically dividing neighbours. In neuroblasts, Serine180 phosphomutants disrupt the timing of basal polarisation. Serine180 phosphomutants also affect the specification and binary cell fate determination of sensory organ precursors as well as Baz localisation during their asymmetric cell divisions. Finally, we show that CDK1 phosphorylates Serine-S180 and an equivalent Serine on human PAR-3 in vitro.

Abstract 372: Cell polarity proteins as novel regulators of macropinocytosis in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is considered one of the deadliest cancers. This is likely because it is detected at a very late stage and the therapeutic approaches that have been used for its treatment have proven to be quite ineffective. Therefore, new therapeutic strategies are needed for a better prognosis in patients suffering of pancreatic cancer. PDAC cells often rewire their metabolism to survive and progress. This metabolic rewiring permits the acquisition of nutrients by the cells when these are exposed to a poor nutrient microenvironment. One of the most used processes in nutrient stress conditions is the “cell-drinking” pathway macropinocytosis, an endocytic pathway that cancer cells exploit to support cancer metabolism in such unfavorable conditions. Macropinocytosis inhibition has shown to suppress PDAC growth in mice and thus, it has become a promising therapeutic target for PDAC treatment. Here, we describe that atypical Protein Kinase C (aPKC) isoforms Protein Kinase C zeta (PKCζ) and Protein Kinase C iota (PKCι) regulate PDAC macropinocytosis in the context of glutamine stress. Genetic depletion and pharmacological inhibition of aPKC impair macropinocytosis promotion in PDAC cells exposed to a glutamine-deprived environment and in cells treated with DON, a glutamine analog that blocks glutamine metabolism. In epithelial cells, aPKCs are known to regulate the establishment of cell polarity in association with the scaffold proteins Par3 and Par6, constituting the cell polarity complex, which controls the function of different targets such as Par1 kinases. Loss of Par3, Par6 and Par-1a affects glutamine stress associated macropinocytosis in a similar extent than aPKC depletion, presenting a novel role of the cell polarity protein network in the modulation of macropinocytic uptake. Citation Format: Guillem Lambies Barjau, Szu-Wei Lee, Cosimo Commisso. Cell polarity proteins as novel regulators of macropinocytosis in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 372.

Impact of fine particulate matter on liver injury: evidence from human, mice and cells

BackgroundA recently discovered risk factor for chronic liver disease is ambient fine particulate matter (PM2.5). Our research aims to elucidate the effects of PM2.5 on liver injury and the potential molecular mechanisms. Methods and resultsA population-based longitudinal study involving 102,918 participants from 15 Chinese cities, using linear mixed-effect models, found that abnormal alterations in liver function were significantly associated with long-term exposure to PM2.5. The serum levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, direct bilirubin, and triglyceride increased by 2.05%, 2.04%, 0.58%, 2.99%, and 1.46% with each 10 µg/m3 increase in PM2.5. In contrast, the serum levels of total protein, albumin, and prealbumin decreased by 0.27%, 0.48%, and 2.42%, respectively. Mice underwent chronic inhalation exposure to PM2.5 experienced hepatic inflammation, steatosis and fibrosis. In vitro experiments found that hepatocytes experienced an inflammatory response and lipid metabolic dysregulation due to PM2.5, which also activated hepatic stellate cells. The down-regulation and mis-localization of polarity protein Par3 mediated PM2.5-induced liver injury. ConclusionsPM2.5 exposure induced liver injury, mainly characterized by steatosis and fibrosis. The down-regulation and mis-localization of Par3 were important mechanisms of liver injury induced by PM2.5.

BET Inhibition Rescues Acinar-Ductal-Metaplasia and Ciliogenesis and Ameliorates Chronic Pancreatitis-Driven Changes in Mice With Loss of the Polarity Protein Par3

The apical-basal polarity of pancreatic acinar cells is essential for maintaining tissue architecture. However, the mechanisms by which polarity proteins regulate acinar pancreas injury and regeneration are poorly understood. Cerulein-induced pancreatitis was induced in mice with conditional deletion of the polarity protein Par3 in the pancreas. The impact of Par3 loss on pancreas injury and regeneration was assessed by histologic analyses and transcriptional profiling by RNA sequencing. Mice were pretreated with the bromodomain and extraterminal domain (BET) inhibitor JQ1 before cotreatment with cerulein to determine the effect of BET inhibition on pancreas injury and regeneration. Initially, we show that Par3 is increased in acinar-ductal metaplasia (ADM) lesions present in human and mouse chronic pancreatitis specimens. Although Par3 loss disrupts tight junctions, Par3 is dispensable for pancreatogenesis. However, with aging, Par3 loss results in low-grade inflammation, acinar degeneration, and pancreatic lipomatosis. Par3 loss exacerbates acute pancreatitis-induced injury and chronic pancreatitis-induced acinar cell loss, promotes pancreatic lipomatosis, and prevents regeneration. Par3 loss also results in suppression of chronic pancreatitis-induced ADM and primary ciliogenesis. Notably, targeting BET proteins attenuates chronic pancreatitis-induced loss of primary cilia and promotes ADM in mice lacking pancreatic Par3. Targeting BET proteins also attenuates cerulein-induced acinar cell loss and enhances recovery of acinar cell mass and body weight of mice lacking pancreatic Par3. Combined, this study demonstrates how Par3 restrains chronic pancreatitis-induced changes in the pancreas and identifies a potential role for BET inhibitors to attenuate pancreas injury and facilitate regeneration.

Claudin-3 in the non-neural ectoderm is essential for neural fold fusion in chicken embryos

The neural tube, the embryonic precursor to the brain and spinal cord, begins as a flat sheet of epithelial cells, divided into non-neural and neural ectoderm. Proper neural tube closure requires that the edges of the neural ectoderm, the neural folds, to elevate upwards and fuse along the dorsal midline of the embryo. We have previously shown that members of the claudin protein family are required for the early phases of chick neural tube closure. Claudins are transmembrane proteins, localized in apical tight junctions within epithelial cells where they are essential for regulation of paracellular permeability, strongly involved in apical-basal polarity, cell-cell adhesion, and bridging the tight junction to cytoplasmic proteins. Here we explored the role of Claudin-3 (Cldn3), which is specifically expressed in the non-neural ectoderm. We discovered that depletion of Cldn3 causes folic acid-insensitive primarily spinal neural tube defects due to a failure in neural fold fusion. Apical cell surface morphology of Cldn3-depleted non-neural ectodermal cells exhibited increased membrane blebbing and smaller apical surfaces. Although apical-basal polarity was retained, we observed altered Par3 and Pals1 protein localization patterns within the apical domain of the non-neural ectodermal cells in Cldn3-depleted embryos. Furthermore, F-actin signal was reduced at apical junctions. Our data presents a model of spina bifida, and the role that Cldn3 is playing in regulating essential apical cell processes in the non-neural ectoderm required for neural fold fusion.

Membrane extraction in native lipid nanodiscs reveals dynamic regulation of Cdc42 complexes during cell polarization

Embryonic development requires the establishment of cell polarity to enable cell fate segregation and tissue morphogenesis. This process is regulated by Par complex proteins, which partition into polarized membrane domains and direct downstream polarized cell behaviors. The kinase aPKC (along with its cofactor Par6) is a key member of this network and can be recruited to the plasma membrane by either the small GTPase Cdc42 or the scaffolding protein Par3. Although in vitro interactions among these proteins are well established, much is still unknown about the complexes they form during development. Here, to enable the study of membrane-associated complexes ex vivo, we used a maleic acid copolymer to rapidly isolate membrane proteins from single C. elegans zygotes into lipid nanodiscs. We show that native lipid nanodisc formation enables detection of endogenous complexes involving Cdc42, which are undetectable when cells are lysed in detergent. We found that Cdc42 interacts more strongly with aPKC/Par6 during polarity maintenance than polarity establishment, two developmental stages that are separated by only a few minutes. We further show that Cdc42 and Par3 do not bind aPKC/Par6 simultaneously, confirming recent in vitro findings in an ex vivo context. Our findings establish a new tool for studying membrane-associated signaling complexes and reveal an unexpected mode of polarity regulation via Cdc42.

Cell polarity: Adapting the PAR cascade to diverse cellular contexts

Two new studies shed light on the intricacies of Caenorhabditis elegans embryo patterning, revealing how the conserved interaction and crosstalk of PAR proteins are adapted to perceive distinct cues, ultimately shaping unique asymmetries in form and function.

Loss of polarity protein Par3 in the intestinal epithelium promotes colitis-associated colorectal cancer progression by damaging tight junction assembly.

Partitioning defective 3 (Par3) is a polarity protein critical in establishing epithelial cell polarity and tight junctions (TJs). Impaired intestinal epithelial barrier integrity is closely associated with colitis-associated colorectal cancer (CRC) progression. According to the GEO and TCGA database analyses, we first observed that the expression of Par3 was reduced in CRC patients. To understand how Par3 is related to CRC, we investigated the role of Par3 in the development of CRC using an in vivo genetic approach. Our results show that the intestinal epithelium-specific PAR3 deletion mice demonstrated a more severe CRC phenotype in the context of azoxymethane/dextran sodium sulfate (AOM/DSS) treatment, with a corresponding increase in tumor number and inflammatory cytokines profile. Mechanistically, loss of Par3 disrupts the TJs of the intestinal epithelium and increases mucosal barrier permeability. The interaction of Par3 with ZO-1 prevents intramolecular interactions within ZO-1 protein and facilitates the binding of occludin to ZO-1, hence preserving TJs integrity. Our results suggest that Par3 deficiency permits pathogenic bacteria and their endotoxins to penetrate the intestinal submucosa and activate TLR4/MyD88/NF-κB signaling, promoting inflammation-driven CRC development and that Par3 may be a novel potential molecular marker for the diagnosis of early-stage CRC.

Nox1-based NADPH oxidase regulates the Par protein complex activity to control cell polarization.

Cell migration is essential for many biological and pathological processes. Establishing cell polarity with a trailing edge and forming a single lamellipodium at the leading edge of the cell is crucial for efficient directional cell migration and is a hallmark of mesenchymal cell motility. Lamellipodia formation is regulated by spatial-temporal activation of the small GTPases Rac and Cdc42at the front edge, and RhoA at the rear end. At a molecular level, partitioning-defective (Par) protein complex comprising Par3, Par6, and atypical Protein Kinase (aPKC isoforms ζ and λ/ι) regulates front-rear axis polarization. At the front edge, integrin clustering activates Cdc42, prompting the formation of Par3/Par6/aPKC complexes to modulate MTOC positioning and microtubule stabilization. Consequently, the Par3/Par6/aPKC complex recruits Rac1-GEF Tiam to activate Rac1, leading to lamellipodium formation. At the rear end, RhoA-ROCK phosphorylates Par3 disrupting its interaction with Tiam and inactivating Rac1. RhoA activity at the rear end allows the formation of focal adhesions and stress fibers necessary to generate the traction forces that allow cell movement. Nox1-based NADPH oxidase is necessary for PDGF-induced migration in vitro and in vivo for many cell types, including fibroblasts and smooth muscle cells. Here, we report that Nox1-deficient cells failed to acquire a normal front-to-rear polarity, polarize MTOC, and form a single lamellipodium. Instead, these cells form multiple protrusions that accumulate Par3 and active Tiam. The exogenous addition of H2O2 rescues this phenotype and is associated with the hyperactivation of Par3, Tiam, and Rac1. Mechanistically, Nox1 deficiency induces the inactivation of PP2A phosphatase, leading to increased activation of aPKC. These results were validated in Nox1y/- primary mouse aortic smooth muscle cells (MASMCs), which also showed PP2A inactivation after PDGF-BB stimulation consistent with exacerbated activation of aPKC. Moreover, we evaluated the physiological relevance of this signaling pathway using a femoral artery wire injury model to generate neointimal hyperplasia. Nox1y/- mice showed increased staining for the inactive form of PP2A and increased signal for active aPKC, suggesting that PP2A and aPKC activities might contribute to reducing neointima formation observed in the arteries of Nox1y/- mice.

Persistent cell contacts enable E-cadherin/HMR-1- and PAR-3-based symmetry breaking within a developing C. elegans epithelium

Tissue-wide patterning is essential to multicellular development, requiring cells to individually generate polarity axes and coordinate them in space and time with neighbors. Using the C.elegans intestinal epithelium, we identified a patterning mechanism that is informed by cell contact lifetime asymmetry and executed via the scaffolding protein PAR-3 and the transmembrane protein E-cadherin/HMR-1. Intestinal cells break symmetry as PAR-3 and HMR-1 recruit apical determinants into punctate "local polarity complexes" (LPCs) at homotypic contacts. LPCs undergo an HMR-1-based migration to a common midline, thereby establishing tissue-wide polarity. Thus, symmetry breaking results from PAR-3-dependent intracellular polarization coupled to HMR-1-based tissue-level communication, which occurs through a non-adhesive signaling role for HMR-1. Differential lifetimes between homotypic and heterotypic cell contacts are created by neighbor exchanges and oriented divisions, patterning where LPCs perdure and thereby breaking symmetry. These cues offer a logical and likely conserved framework for how epithelia without obvious molecular asymmetries can polarize.

Involvement of PAR2 in inflammatory mediator release from human blood eosinophils

Proteinase Activated Receptors (PARs) are the members of G-protein-coupled receptor family and can be cleaved by certain serine proteases to expose a tethered ligand domain, which binds and activates the receptors to initiate multiple signaling cascades. There is some evidence that certain proteases may regulate target cells by activating PARs. There are many studies, in which PARs play important roles in inflammation. One study indicated that PAR2 inhibition and deletion significantly suppressed the degree of inflammation due to decreased IL-6 and IL-1 levels. Another study also showed that PARs activation could mediate reactive oxygen species production and MAPK signaling leading to alveolar inflammation. In addition, platelet-derived CAPN1 can trigger the vascular inflammation associated with diabetes via cleavage of PAR1 and the release of TNF from the endothelial cell surface, and sarsasapogenin may alleviate diabetic nephropathy by the downregulation of PAR1. Another Phellodendron amurense bark extract can suppress the particulate matter-induced Ca2+ influx caused by direct action upon PAR2, alleviating inflammation and maintaining homeostatic levels of cell adhesion components. There are also other two antagonists of I-287 and GB88, which can reduce the PAR2-mediated inflammatory reaction. In this study, we tested expression of PARs and IL-5, IL-6, RANTES and ECP release from human blood eosinophils using different enzymes and PAR agonists. The expression of PARs was assessed in human blood eosinophils by flow cytometry and RT-PCR, and the levels of cytokine and eosinophil cationic protein (ECP) in the cultured supernatants were determined with ELISA kits. Flow cytometry shows that human eosinophils express PAR2 protein and do not express PAR1, PAR3 and PAR4 proteins. RT-PCR analysis revealed expression of PAR2 and PAR3 genes in human eosinophils. Tryptase, trypsin and elastase can induce significant IL-5, IL-6 and ECP release. Trypsin and elastase may also stimulate RANTES secretion, but tryptase cannot induce the RANTES secretion. Tryptase, trypsin and elastase-induced cytokine and ECP release from human blood eosinophils most likely occurs via activation of PAR2.

Synthesis of Novel Dual Target Inhibitors of CDK12 and PARP1 and Their Antitumor Activities in HER2-Positive Breast Cancers.

Several anti-human epidermal growth factor receptor 2 (HER2) treatments have improved the landscape of HER2-positive breast cancer (BC) over the past few years; due to the heterogeneity of the disease itself, the drug resistance mechanisms and relapse are still the main issue in HER2-positive BC. Here, we intended to target simultaneous inhibition of both poly ADP-ribose polymerase 1 (PARP1) and cyclin-dependent kinase 12 (CDK12) that have had an impact on this disease up to their implementation in clinical practice. We successfully screened PARP1 inhibitors (PARPis) containing bicyclic tetrahydropyridine pyrimidines with antitumor activity. Most synthesized compounds with various alcohols were more effective at killing tumor cells than olaparib (ola), especially in HER2-positive cancer cells. Among them, compound 9 showed potent inhibitory effects on PARP1 enzymatic activity and the PAR protein level; moreover, the expression of CDK12 was inhibited by compound 9. Overall, compound 9 exhibited a significant antitumor effect by inhibiting DNA damage repair in tumors.

Ccdc85c-Par3 condensates couple cell polarity with Notch to control neural progenitor proliferation

Polarity proteins regulate the proliferation and differentiation of neural progenitors to generate neurons during brain development through multiple signaling pathways. However, how cell polarity couples the signaling pathways remains unclear. Here, we show that coiled-coil domain-containing protein 85c (Ccdc85c) interacts with the polarity protein Par3 to regulate the proliferation of radial glial cells (RGCs) via phase separation coupled to percolation (PSCP). We find that the interaction with Ccdc85c relieves the intramolecular auto-inhibitionof Par3, which leads to PSCP of Par3. Downregulation of Ccdc85c causes RGC differentiation. Importantly, the open conformation of Par3 facilitates the recruitment of the Notch regulator Numb to the Par3 condensates, which might prevent the attenuation of Notch activity to maintain RGC proliferation. Furthermore, ectopic activation of Notch signaling rescues RGC proliferation defects caused by the downregulation of Ccdc85c. These results suggest that Ccdc85c-mediated PSCP of Par3 regulates Notch signaling to control RGC proliferation during brain development.

Design principles for selective polarization of PAR proteins by cortical flows.

Clustering of membrane-associated molecules is thought to promote interactions with the actomyosin cortex, enabling size-dependent transport by actin flows. Consistent with this model, in the Caenorhabditis elegans zygote, efficient anterior segregation of the polarity protein PAR-3 requires oligomerization. However, through direct assessment of local coupling between motion of PAR proteins and the underlying cortex, we find no links between PAR-3 oligomer size and the degree of coupling. Indeed, both anterior and posterior PAR proteins experience similar advection velocities, at least over short distances. Consequently, differential cortex engagement cannot account for selectivity of PAR protein segregation by cortical flows. Combining experiment and theory, we demonstrate that a key determinant of differential segregation of PAR proteins by cortical flow is the stability of membrane association, which is enhanced by clustering and enables transport across cellular length scales. Thus, modulation of membrane binding dynamics allows cells to achieve selective transport by cortical flows despite widespread coupling between membrane-associated molecules and the cell cortex.

Abstract B061: Updated analysis of “CANCAP03” – A study into the pharmacodynamic biomarker effects of olaparib (PARP inhibitor) ± degarelix (GnRH antagonist) given prior to radical prostatectomy

Abstract Background: PARP inhibition with androgen deprivation is an effective treatment for Prostate Cancer (PC). We investigated the effects of a two-week course of olaparib±degarelix in the “window” before radical prostatectomy (RP). Methods: Primary endpoint: level of PARP inhibition. Secondary endpoints: safety and feasibility of the trial design. Exploratory endpoints: changes in relevant biological pathways after olaparib±degarelix. Participants (Pt) with localised PC (D’Amico intermediate or high risk) were randomly allocated (1:1) to receive olaparib 300mg bid for two weeks ± degarelix (240mg s/c). Recruitment target was twenty evaluable Pts. Compliance (patient diaries) and toxicity (CTCAE v4.03) were assessed. Diagnostic biopsies with subsequent prostatectomy samples were analysed by IHC for PAR protein expression and whole transcriptome gene expression analysis was performed using NovaSeq. Gene mutation profiling was performed using a custom 350 gene panel, sequenced on Illumina NextSeq 2000 and analysed with in-house bioinformatics pipeline. Serum samples for PSA and testosterone were taken at baseline, day 15 and 42 post RP. Results: 24 patients were recruited, 23 evaluable for safety and 20 for primary endpoint (11 olaparib, 9 olaparib+degarelix). All related AE ≤ Grade (Gr) 2, majority Gr 1. Commonest AEs: fatigue, nausea & vomiting (olaparib) or injection site reaction, fatigue & nausea (olaparib+degarelix). No RP was delayed by an AE. PSA declined from baseline in all (8/8) Pt after olaparib+degarelix and 3/9 Pt after olaparib (-11, -17 & -47%). PAR protein expression decreased in both cohorts after treatment, with the mean reduction in PAR expression olaparib > olaparib+degarelix (p=0.01). After olaparib, Pt with greatest PSA change also had greatest reduction in PAR expression compared with other Pt (p=0.008); no correlation between PSA and PAR change was detected in olaparib+degarelix Pt. Pathogenic mutations (in BRCA2/ATM and ATM) were detected in two Pt. Upregulation of gene signatures, including homologous recombination signature, were noted following combined olaparib+degarelix treatment. Conclusions: We have previously reported that toxicities were predictable and manageable, with no drug-related delay in RP for Pt. Updated results confirm PARP inhibition (reduced PAR expression) in PC samples after olaparib and (to a lesser degree) olaparib+degarelix. Up-regulation of homologous recombination deficiency gene signature occurred after olaparib+degarelix. Further assessments of post-treatment tissues are ongoing to establish mechanisms of action of combination therapy across all genetic backgrounds. Citation Format: Harveer Dev, Mark Linch, Howard Kynaston, Greg Shaw, Krishna Narahari, Tatiana Hernández, Anne Warren, Alopa Malaviya, Vincent Gnanapragasam, Elizabeth Harrington, Niedzica Camacho, Silvia Glont, Massimo Sqautrito, Joshua Armenia, Luiza Moore, Robert Hanson, Toby Milne-Clarke, Shubha Anand, Charlie Massie, Nimish Shah, Simon Pacey. Updated analysis of “CANCAP03” – A study into the pharmacodynamic biomarker effects of olaparib (PARP inhibitor) ± degarelix (GnRH antagonist) given prior to radical prostatectomy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B061.