Feed-forward Loop Research Articles

Prmt6 represses the pro-adipogenic Ppar-gamma–C/ebp-alpha transcription factor loop

The feed-forward loop between the transcription factors Ppar-gamma and C/ebp-alpha is critical for lineage commitment during adipocytic differentiation. Ppar-gamma interacts with epigenetic cofactors to activate C/ebp-alpha and the downstream adipocytic gene expression program. Therefore, knowledge of the epigenetic cofactors associated with Ppar-gamma, is central to understanding adipocyte differentiation in normal differentiation and disease. We found that Prmt6 is present with Ppar-gamma on the Ppar-gamma and C/ebp-alpha promoter. It contributes to the repression of C/ebp-alpha expression, in part through its ability to induce H3R2me2a. During adipocyte differentiation, Prmt6 expression is reduced and the methyltransferase leaves the promoters. As a result, the expression of Ppar-gamma and C/ebp-alpha is upregulated and the adipocytic gene expression program is established. Inhibition of Prmt6 by a small molecule enhances adipogenesis, opening up the possibility of epigenetic manipulation of differentiation. Our data provide detailed information on the molecular mechanism controlling the Ppar-gamma–C/ebp-alpha feed-forward loop. Thus, they advance our understanding of adipogenesis in normal and aberrant adipogenesis.

Stability enhancement control strategy for grid-connected wind power system based on composite feedforward damping

The stability of grid-connected wind power system (GCWPS) is prone to deteriorate due to the impedance interaction between wind turbines and the weak grid. For purpose of finding out the cause of power oscillation and effectively improving the stability of GCWPS under weak grid, firstly of all, a frequency coupling impedance model (FCIM) for GCWPS is established. Through analyzing the impedance characteristic of GCWPS, a significant coupling effect between GCWPS impedance and grid impedance in the middle-low frequency region under weak grid is observed, which is easy to cause power oscillation. For purpose of restraining the power oscillation of GCWPS under weak grid, this paper proposes an oscillation suppression strategy based on composite feedforward damping. Virtual damping is incorporated into the feedforward loop to rectify the impedance characteristic curve of GCWPS. Then, a FCIM of GCWPS that adopts the proposed oscillation suppression strategy is established, and the stability analysis demonstrates that the proposed strategy effectively enhances the GCWPS stability under weak grid. Additionally, compared with conventional control strategy, GCWPS exhibits enhanced adaptability to the increased output power and phase-locked loop (PLL) bandwidth under weak grid. Finally, experiments are undertaken to evidence the feasibility of the proposed oscillation suppression strategy.

The Discovery of 7-Isopropoxy-2-(1-methyl-2-oxabicyclo[2.1.1]hexan-4-yl)-N-(6-methylpyrazolo[1,5-a]pyrimidin-3-yl)imidazo[1,2-a]pyrimidine-6-carboxamide (BIO-7488), a Potent, Selective, and CNS-Penetrant IRAK4 Inhibitor for the Treatment of Ischemic Stroke.

Interleukin receptor-associated kinase 4 (IRAK4) is a key node of signaling within the innate immune system that regulates the production of inflammatory cytokines and chemokines. The presence of damage-associated molecular patterns (DAMPs) after tissue damage such as stroke or traumatic brain injury (TBI) initiates signaling through the IRAK4 pathway that can lead to a feed-forward inflammatory loop that can ultimately hinder patient recovery. Herein, we describe the first potent, selective, and CNS-penetrant IRAK4 inhibitors for the treatment of neuroinflammation. Lead compounds from the series were evaluated in CNS PK/PD models of inflammation, as well as a mouse model of ischemic stroke. The SAR optimization detailed within culminates in the discovery of BIO-7488, a highly selective and potent IRAK4 inhibitor that is CNS penetrant and has excellent ADME properties.

ADAMTS4 is a crucial proteolytic enzyme for versican cleavage in the amnion at parturition

Hyalectan cleavage may play an important role in extracellular matrix remodeling. However, the proteolytic enzyme responsible for hyalectan degradation for fetal membrane rupture at parturition remains unknown. Here, we reveal that versican (VCAN) is the major hyalectan in the amnion, where its cleavage increases at parturition with spontaneous rupture of membrane. We further reveal that ADAMTS4 is a crucial proteolytic enzyme for VCAN cleavage in the amnion. Inflammatory factors may enhance VCAN cleavage by inducing ADAMTS4 expression and inhibiting ADAMTS4 endocytosis in amnion fibroblasts. In turn, versikine, the VCAN cleavage product, induces inflammatory factors in amnion fibroblasts, thereby forming a feedforward loop between inflammation and VCAN degradation. Mouse studies show that intra-amniotic injection of ADAMTS4 induces preterm birth along with increased VCAN degradation and proinflammatory factors abundance in the fetal membranes. Conclusively, there is enhanced VCAN cleavage by ADAMTS4 in the amnion at parturition, which can be reenforced by inflammation.

Lupus autoantibodies initiate neuroinflammation sustained by continuous HMGB1:RAGE signaling and reversed by increased LAIR-1 expression.

Cognitive impairment is a frequent manifestation of neuropsychiatric systemic lupus erythematosus, present in up to 80% of patients and leading to a diminished quality of life. In the present study, we used a model of lupus-like cognitive impairment that is initiated when antibodies that crossreact with excitatory neuronal receptors penetrate the hippocampus, causing immediate, self-limited, excitotoxic death of hippocampal neurons, which is then followed by a significant loss of dendritic complexity in surviving neurons. This injury creates a maladaptive equilibrium that is sustained in mice for at least 1 year. We identified a feedforward loop of microglial activation and microglia-dependent synapse elimination dependent on neuronal secretion of high mobility group box 1 protein (HMGB1) which binds the receptor for advanced glycation end products (RAGE) and leads to microglial secretion of C1q, upregulation of interleukin-10 with consequent downregulation of leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), an inhibitory receptor for C1q. Treatment with a centrally acting angiotensin-converting enzyme inhibitor or with an angiotensin-receptor blocker restored a healthy equilibrium, microglial quiescence and intact spatial memory.

Pancreatic Cancer Treatment Targeting the HGF/c-MET Pathway: The MEK Inhibitor Trametinib.

Pancreatic cancer is characterized by fibrosis/desmoplasia in the tumor microenvironment, which is primarily mediated by pancreatic stellate cells and cancer-associated fibroblasts. HGF/c-MET signaling, which is instrumental in embryonic development and wound healing, is also implicated for its mitogenic and motogenic properties. In pancreatic cancer, this pathway, along with its downstream signaling pathways, is associated with disease progression, prognosis, metastasis, chemoresistance, and other tumor-related factors. Other features of the microenvironment in pancreatic cancer with the HGF/c-MET pathway include hypoxia, angiogenesis, metastasis, and the urokinase plasminogen activator positive feed-forward loop. All these attributes critically influence the initiation, progression, and metastasis of pancreatic cancer. Therefore, targeting the HGF/c-MET signaling pathway appears promising for the development of innovative drugs for pancreatic cancer treatment. One of the primary downstream effects of c-MET activation is the MAPK/ERK (Ras, Ras/Raf/MEK/ERK) signaling cascade, and MEK (Mitogen-activated protein kinase kinase) inhibitors have demonstrated therapeutic value in RAS-mutant melanoma and lung cancer. Trametinib is a selective MEK1 and MEK2 inhibitor, and it has evolved as a pivotal therapeutic agent targeting the MAPK/ERK pathway in various malignancies, including BRAF-mutated melanoma, non-small cell lung cancer and thyroid cancer. The drug's effectiveness increases when combined with agents like BRAF inhibitors. However, resistance remains a challenge, necessitating ongoing research to counteract the resistance mechanisms. This review offers an in-depth exploration of the HGF/c-MET signaling pathway, trametinib's mechanism, clinical applications, combination strategies, and future directions in the context of pancreatic cancer.

Abstract B010: PITAR, a p53 inactivating TRIM28 associated RNA, executes an alternate way of p53 inactivation

Abstract B010: PITAR, a <i>p</i>53 inactivating TRIM28 associated RNA, executes an alternate way of p53 inactivation

Incoherent feedforward loop dominates the robustness and tunability of necroptosis biphasic, emergent, and coexistent dynamics

Biphasic dynamics, the variable-dependent ability to enhance or restrain biological function, is prevalent in natural systems. Accompanied by biphasic dynamics, necroptosis signaling also appears emergent and coexistent dynamics. However, it remains elusive how the properties of these dynamics are characterized by specific circuit structures and components. Starting with necroptosis circuit modeling, we systematically analyzed the network topology for achieving RIP1-dependent biphasic, emergent, and coexistent (BEC) dynamics. RIP1-RIP3-Caspase-8 (C8) incoherent feedforward loop embedded with positive feedback of RIP3 to RIP1 is identified as the core topology. The peak value of RIP3 phosphorylation is determined to present a scale-invariant feature, dictating BEC dynamics and the bell-shaped regulation of necroptosis biphasic dynamics. To quantitatively determine the uncertainty of necroptosis coexistent dynamics, potential landscape and Shannon entropy that measure entropy production during cell death are introduced for the first time. Further random necroptosis circuit analysis identifies the bell-shaped regulation of necroptosis biphasic dynamics by RIP3 auto-phosphorylation, which acts as a complementary process for robustly attaining BEC dynamics. Finally, we searched all possible two- and three-node circuit topologies to screen those that could perform BEC dynamics. A complete atlas of three-node circuit BEC dynamics is generated and only three minimal circuits emerge as robust solutions, confirming incoherent feedforward loop is the core topology. Analysis of the association between the minimal circuit structure and robustness proves that the identified optimal functional achievement structure is highly consistent with the experimental observed RIP1-RIP3-C8 topology. Overall, through highlighting a finite set of circuits, this study yields guiding principles that enable the mapping, modulation, and design of circuits for BEC dynamics in diverse synthetic biology applications.

Pathogenic GATA2 genetic variants utilize an obligate enhancer mechanism to distort a multilineage differentiation program

Mutations in genes encoding transcription factors inactivate or generate ectopic activities to instigate pathogenesis. By disrupting hematopoietic stem/progenitor cells, GATA2 germline variants create a bone marrow failure and leukemia predisposition, GATA2 deficiency syndrome, yet mechanisms underlying the complex phenotypic constellation are unresolved. We used a GATA2-deficient progenitor rescue system to analyze how genetic variation influences GATA2 functions. Pathogenic variants impaired, without abrogating, GATA2-dependent transcriptional regulation. Variants promoted eosinophil and repressed monocytic differentiation without regulating mast cell and erythroid differentiation. While GATA2 and T354M required the DNA-binding C-terminal zinc finger, T354M disproportionately required the N-terminal finger and N terminus. GATA2 and T354M activated a CCAAT/Enhancer Binding Protein-ε (C/EBPε) enhancer, creating a feedforward loop operating with the T-cell Acute Lymphocyte Leukemia-1 (TAL1) transcription factor. Elevating C/EBPε partially normalized hematopoietic defects of GATA2-deficient progenitors. Thus, pathogenic germline variation discriminatively spares or compromises transcription factor attributes, and retaining an obligate enhancer mechanism distorts a multilineage differentiation program.

Biopterin metabolism and nitric oxide recoupling in cancer.

Tetrahydrobiopterin is a cofactor necessary for the activity of several enzymes, the most studied of which is nitric oxide synthase. The role of this cofactor-enzyme relationship in vascular biology is well established. Recently, tetrahydrobiopterin metabolism has received increasing attention in the field of cancer immunology and immunotherapy due to its involvement in the cytotoxic T cell response. Past research has demonstrated that when the availability of BH4 is low, as it is in chronic inflammatory conditions and tumors, electron transfer in the active site of nitric oxide synthase becomes uncoupled from the oxidation of arginine. This results in the production of radical species that are capable of a direct attack on tetrahydrobiopterin, further depleting its local availability. This feedforward loop may act like a molecular switch, reinforcing low tetrahydrobiopterin levels leading to altered NO signaling, restrained immune effector activity, and perpetual vascular inflammation within the tumor microenvironment. In this review, we discuss the evidence for this underappreciated mechanism in different aspects of tumor progression and therapeutic responses. Furthermore, we discuss the preclinical evidence supporting a clinical role for tetrahydrobiopterin supplementation to enhance immunotherapy and radiotherapy for solid tumors and the potential safety concerns.

CHOP-mediated Gasdermin E expression promotes pyroptosis, inflammation, and mitochondrial damage in renal ischemia-reperfusion injury

In clinical practice, renal ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI), often leading to acute renal failure or end-stage renal disease (ESRD). The current understanding of renal IRI mechanisms remains unclear, and effective therapeutic strategies and clear targets are lacking. Therefore, the need to find explicit and effective ways to reduce renal IRI remains a scientific challenge. The current study explored pyroptosis, a type of inflammation-regulated programmed cell death, and the role of Gasdermins E (GSDME)-mediated pyroptosis, mitochondrial damage, and inflammation in renal IRI. The analysis of human samples showed that the expression levels of GSDME in normal human renal tissues were higher than those of GSDMD. Moreover, our study demonstrated that GSDME played an important role in mediating pyroptosis, inflammation, and mitochondrial damage in renal IRI. Subsequently, GSDME-N accumulated in the mitochondrial membrane, leading to mitochondrial damage and activation of caspase3, which generated a feed-forward loop of self-amplification injury. However, GSDME knockout resulted in the amelioration of renal IRI. Moreover, the current study found that the transcription factor CHOP was activated much earlier in renal IRI. Inhibition of BCL-2 by CHOP leaded to casapse3 activation, resulting in mitochondrial damage and apoptosis; not only that, but CHOP positively regulated GSDME thereby causing pyroptosis. Therefore, this study explored the transcriptional mechanisms of GSDME during IRI development and the important role of CHOP/Caspase3/GSDME mechanistic axis in regulating pyroptosis in renal IRI. This axis might serve as a potential therapeutic target.

Orthogonal inducible control of Cas13 circuits enables programmable RNA regulation in mammalian cells.

RNA plays an indispensable role in mammalian cell functions. Cas13, a class of RNA-guided ribonuclease, is a flexible tool for modifying and regulating coding and non-coding RNAs, with enormous potential for creating new cell functions. However, the lack of control over Cas13 activity has limited its cell engineering capability. Here, we present the CRISTAL (Control of RNA with Inducible SpliT CAs13 Orthologs and Exogenous Ligands) platform. CRISTAL is powered by a collection (10 total) of orthogonal split inducible Cas13 effectors that can be turned ON or OFF via small molecules in multiple cell types, providing precise temporal control. Also, we engineer Cas13 logic circuits that can respond to endogenous signaling and exogenous small molecule inputs. Furthermore, the orthogonality, low leakiness, and high dynamic range of our inducible Cas13d and Cas13b enable the design and construction of a robust incoherent feedforward loop, leading to near-perfect and tunable adaptation response. Finally, using our inducible Cas13 effectors, we achieve simultaneous multiplexed control of multiple genes in vitro and in mice. Together, our CRISTAL design represents a powerful platform for precisely regulating RNA dynamics to advance cell engineering and elucidate RNA biology.

The local topology of dynamical network models for biology

Abstract The search for motifs—recurrent patterns in network topology—has led to the identification of universal classes of complex systems across diverse fields and has served as a quantitative tool to reveal common properties in both evolved and designed networks. In this study, we investigate the presence and significance of network superfamilies—previously identified through the census of triadic motifs—in the largest data set of dynamic, biological network models. We present triad significance profiles of 71 existing biological network models, all experimentally inspired. The generated data are treated in an unbiased manner and consistently clustered into two classes using several unsupervised techniques. The more prevalent class does exhibit a strong correlation with the superfamily of sensory transmission networks, which are characterized by the feedforward loop motif commonly found in signal-processing systems. Surprisingly, the remaining class shows a better correlation with the superfamily of word-adjacency networks. To better understand this, the results are analysed for varying network size thresholds, and their connection to the effect of model building activity is examined. It is highlighted that the more the model focuses on smaller portions of the regulatory network, as a result of the coarse-graining of the boundary dynamics and the peripheral regions of the network, the more its topology starts resembling that of ‘sentences’ of word-adjacency networks.

Design and Model-Driven Analysis of Synthetic Circuits with the Staphylococcus aureus Dead-Cas9 (sadCas9) as a Programmable Transcriptional Regulator in Bacteria.

Synthetic circuit design is crucial for engineering microbes that process environmental cues and provide biologically relevant outputs. To reliably scale-up circuit complexity, the availability of parts toolkits is central. Streptococcus pyogenes (sp)-derived CRISPR interference/dead-Cas9 (CRISPRi/spdCas9) is widely adopted for implementing programmable regulations in synthetic circuits, and alternative CRISPRi systems will further expand our toolkits of orthogonal components. Here, we showcase the potential of CRISPRi using the engineered dCas9 from Staphylococcus aureus (sadCas9), not previously used in bacterial circuits, that is attractive for its low size and high specificity. We designed a collection of ∼20 increasingly complex circuits and variants in Escherichia coli, including circuits with static function like one-/two-input logic gates (NOT, NAND), circuits with dynamic behavior like incoherent feedforward loops (iFFLs), and applied sadCas9 to fix a T7 polymerase-based cascade. Data demonstrated specific and efficient target repression (100-fold) and qualitatively successful functioning for all circuits. Other advantageous features included low sadCas9-borne cell load and orthogonality with spdCas9. However, different circuit variants showed quantitatively unexpected and previously unreported steady-state responses: the dynamic range, switch point, and slope of NOT/NAND gates changed for different output promoters, and a multiphasic behavior was observed in iFFLs, differing from the expected bell-shaped or sigmoidal curves. Model analysis explained the observed curves by complex interplays among components, due to reporter gene-borne cell load and regulator competition. Overall, CRISPRi/sadCas9 successfully expanded the available toolkit for bacterial engineering. Analysis of our circuit collection depicted the impact of generally neglected effects modulating the shape of component dose-response curves, to avoid drawing wrong conclusions on circuit functioning.

Understanding the impact of risankizumab on keratinocyte-derived IL-23A in a novel organotypic 3D skin model containing IL-23A responsive and IL-17A producing γδ-T-cells

Purpose To study the effects of the anti-IL-23A antibody risankizumab on the IL-36γ/IL-23A/IL-17A signalling cascade we used a newly developed 3D skin model consisting of primary human keratinocytes, fibroblasts and γδ-T-cells. Methods In this in vitro study we developed new full-thickness 3D skin models containing normal human epidermal keratinocytes (NHEK), normal human dermal fibroblasts (NHDF) and IL-23A responsive and IL-17A producing γδ-T-cells. The effects of IL-36γ stimulation with and without risankizumab treatment on IL-23A and IL-17A expression were examined at the RNA and protein levels. Results In preliminary monolayer experiments stimulation of γδ-T-cells with IL-23A promoted the IL-17A expression that was inhibited after risankizumab treatment. Using 3D skin models containing γδ-T-cells, we found that stimulation with IL-36γ significantly increased not only IL-23A but also IL-17A expression. These effects were inhibited by concomitant treatment with risankizumab. Conclusions Our results showed that blockade of IL-23A has inhibitory effects on the IL-36γ/IL-23A feedforward loop. Our newly developed 3D skin model containing IL-23A responsive and IL-17A producing γδ-T-cells enables molecular analysis of targeted therapies aimed at the IL-36γ/IL-23A/IL-17A signalling cascade in psoriasis.

Interplay of miRNA-TF-Gene Through a Novel Six-node Feed-forward Loop Identified Inflammatory Genes as Key Regulators in Type-2 Diabetes

Background: Intricacy in the pathological processes of type 2 diabetes (T2D) invites a need to understand gene regulation at the systems level. However, deciphering the complex gene modulation requires regulatory network construction, Objective: The study aims to construct a six-node feed-forward loop (FFL) to analyze all the diverse inter- and intra- interactions between microRNAs (miRNA) and transcription factors (TF) involved in gene regulation. Methods: The study included 644 genes, 64 TF, and 448 miRNA. A cumulative hypergeometric test was employed to identify the significant miRNA-miRNA and miRNA-TF interaction pairs. In addition, experimentally proven TF-TF pairs were incorporated for the first time in the regulatory network to discern gene regulation. The networks were analyzed to identify crucial genes involved in T2D. Following this, gene ontology was predicted to recognize the biological function that is crucial in T2D. Results: In T2D, the lowest gene regulation for a composite FFL occurs through a four-node FFL variant1 (TF- miRNA-miRNA-Gene, n=14) and the highest regulation via a five-node FFL variant2 (TF-TF-miRNA-Gene, n=353). However, the maximum gene regulation occurs via six-node miRNA FFL (miRNA-miRNA-TF-TF-gene-gene, n=23987). Subnetworks derived from the six-node miRNATF- gene regulatory networks identified interactions among TP53 and NFkB, hsa-miR-125-5p and hsamiR- 155-5p. Conclusion: The core regulation occurs through TP53, NFkB, hsa-miR-125-5p, and hsa-miR-155-5p FFL implicating the association of inflammation in the pathogenesis of T2D, which occurs majorly via six-node miRNA FFL. Thus regulatory network provides broader insights into the pathogenesis of T2D and can be extended to study the inflammatory mechanisms in various infections.

The Contribution of Innate Immunity in Large-Vessel Vasculitis: Detangling New Pathomechanisms beyond the Onset of Vascular Inflammation.

Large-vessel vasculitis (LVV) are autoimmune and autoinflammatory diseases focused on vascular inflammation. The central core of the intricate immunological and molecular network resides in the disruption of the "privileged immune state" of the arterial wall. The outbreak, initially primed by dendritic cells (DC), is then continuously powered in a feed-forward loop by the intimate cooperation between innate and adaptive immunity. If the role of adaptive immunity has been largely elucidated, knowledge of the critical function of innate immunity in LVV is still fragile. A growing body of evidence has strengthened the active role of innate immunity players and their key signaling pathways in orchestrating the complex pathomechanisms underlying LVV. Besides DC, macrophages are crucial culprits in LVV development and participate across all phases of vascular inflammation, culminating in vessel wall remodeling. In recent years, the variety of potential pathogenic actors has expanded to include neutrophils, mast cells, and soluble mediators, including the complement system. Interestingly, new insights have recently linked the inflammasome to vascular inflammation, paving the way for its potential pathogenic role in LVV. Overall, these observations encourage a new conceptual approach that includes a more in-depth study of innate immunity pathways in LVV to guide future targeted therapies.

Icaritin with autophagy/mitophagy inhibitors synergistically enhances anticancer efficacy and apoptotic effects through PINK1/Parkin-mediated mitophagy in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is among the deadliest malignancies worldwide and still a pressing clinical problem. Icaritin, a natural compound obtained from the Epimedium genus plant, has garnered significant attention as a potential therapeutic drug for HCC therapies. Mitophagy plays a crucial role in mitochondrial quality control through efficiently eliminating damaged mitochondria. However, the specific mechanisms of the interplay between mitophagy and apoptosis in HCC is still unclear. We aimed to explore the cross-talk between icaritin-induced mitophagy and apoptosis in HCC cells and investigate its potential mechanisms. Firstly, we confirmed that icaritin inhibits proliferation and migration while inducing mitochondrial damage and reactive oxygen species (ROS) production in HCC cells. Secondly, based on proteomics analysis, we discovered that icaritin inhibits the growth of tumor cells and disrupts their mitochondrial homeostasis through the regulation of both mitophagy and apoptosis. Thirdly, icaritin causes mitophagy mediated by PINK1-Parkin signaling via regulating feedforward loop. Furthermore, knockdown of PINK1/Parkin leads to inhibition of mitophagy, which promotes cell death induced by icaritin in HCC cells. Finally, autophagy/mitophagy inhibitors remarkably enhance icaritin-induced cell death and anticancer efficacy. Collectively, our findings reveal that icaritin suppresses growth, proliferation and migration of HCC cell through induction of mitophagy and apoptosis, while inhibition of mitophagy significantly increased the anti-cancer and pro-apoptotic effects of icaritin, indicating that targeting autophagy or mitophagy is a novel approach to overcome drug resistance and enhance anticancer therapies.

Abstract A008: Toxic PARP trapping upon cAMP-induced DNA damage reinstates the efficacy of endocrine therapy and CDK4/6 inhibitors in treatment-refractory ER+ breast cancer

Abstract Estrogen receptor (ER)-positive (ER+) breast cancer has the highest incidence rate and accounts for around 75% of all cases. Endocrine therapy and CDK4/6 inhibitors are the standard of care (SOC) in ER+ breast cancer; however, resistance is common, and it greatly reduces patient survival. Therefore, elucidating the mechanisms of sensitivity and resistance to SOC therapy and identifying actionable targets are urgently needed. Here, we show that SOC therapy causes DNA damage and toxic PARP1 trapping upon generation of a functional BRCAness (i.e., BRCA1/2 deficiency) phenotype, leading to increased histone parylation and reduced H3K9 acetylation, resulting in transcriptional blockage and cell death. Mechanistically, SOC therapy downregulates phosphodiesterase 4D (PDE4D), a novel ER target gene in a feedforward loop with ER, resulting in increased cAMP, PKA-dependent phosphorylation of mitochondrial COXIV-I, ROS generation and DNA damage. However, during SOC resistance, an ER-to-EGFR switch induces PDE4D overexpression via c-Jun-mediated transcription. Importantly, we demonstrated that high PDE4D mRNA and protein expression is associated with dramatically worse disease-free survival and overall survival in endocrine therapy-treated ER+ breast cancer. Notably, combining SOC with inhibitors of PDE4D, EGFR or PARP1 overcomes SOC resistance irrespective of the BRCA1/2 status in multiple acquired SOC resistant cell line models, primary cultures and organoids of endocrine resistant ER+ PDXs in vitro and in vivo, providing actionable targets for restoring SOC efficacy. Considering the availability of potent and non-toxic PDE4D inhibitors, clinically approved EGFR and PARP1 inhibitors, our findings have great translational potential. Citation Format: Ozge Saatci, Mein Cetin, Meral Uner, Unal Metin Tokat, Ioulia Chatzistamou, Pelin Gulizar Ersan, Elodie Montaudon, Aytekin Akyol, Sercan Aksoy, Aysegul Uner, Elisabetta Marangoni, Mathew Sajish, Ozgur Sahin. Toxic PARP trapping upon cAMP-induced DNA damage reinstates the efficacy of endocrine therapy and CDK4/6 inhibitors in treatment-refractory ER+ breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A008.

Contribution of membrane-associated oscillators to biological timing at different timescales.

Environmental rhythms such as the daily light-dark cycle selected for endogenous clocks. These clocks predict regular environmental changes and provide the basis for well-timed adaptive homeostasis in physiology and behavior of organisms. Endogenous clocks are oscillators that are based on positive feedforward and negative feedback loops. They generate stable rhythms even under constant conditions. Since even weak interactions between oscillators allow for autonomous synchronization, coupling/synchronization of oscillators provides the basis of self-organized physiological timing. Amongst the most thoroughly researched clocks are the endogenous circadian clock neurons in mammals and insects. They comprise nuclear clockworks of transcriptional/translational feedback loops (TTFL) that generate ∼24h rhythms in clock gene expression entrained to the environmental day-night cycle. It is generally assumed that this TTFL clockwork drives all circadian oscillations within and between clock cells, being the basis of any circadian rhythm in physiology and behavior of organisms. Instead of the current gene-based hierarchical clock model we provide here a systems view of timing. We suggest that a coupled system of autonomous TTFL and posttranslational feedback loop (PTFL) oscillators/clocks that run at multiple timescales governs adaptive, dynamic homeostasis of physiology and behavior. We focus on mammalian and insect neurons as endogenous oscillators at multiple timescales. We suggest that neuronal plasma membrane-associated signalosomes constitute specific autonomous PTFL clocks that generate localized but interlinked oscillations of membrane potential and intracellular messengers with specific endogenous frequencies. In each clock neuron multiscale interactions of TTFL and PTFL oscillators/clocks form a temporally structured oscillatory network with a common complex frequency-band comprising superimposed multiscale oscillations. Coupling between oscillator/clock neurons provides the next level of complexity of an oscillatory network. This systemic dynamic network of molecular and cellular oscillators/clocks is suggested to form the basis of any physiological homeostasis that cycles through dynamic homeostatic setpoints with a characteristic frequency-band as hallmark. We propose that mechanisms of homeostatic plasticity maintain the stability of these dynamic setpoints, whereas Hebbian plasticity enables switching between setpoints via coupling factors, like biogenic amines and/or neuropeptides. They reprogram the network to a new common frequency, a new dynamic setpoint. Our novel hypothesis is up for experimental challenge.