Tolerance Pathways Research Articles

Unraveling the signaling pathways of phytohormones underlying salt tolerance in Elymus sibiricus: A transcriptomic and metabolomic approach

Understanding phytohormonal signaling is crucial for elucidating plant defense mechanisms against environmental stressors. However, knowledge regarding phytohormone-mediated tolerance pathways under salt stress in Elymus sibiricus, an important species for forage and ecological restoration, remains limited. In this study, transcriptomic and metabolomic approaches uncover the dynamics of phytohormonal signaling in Elymus sibiricus under salt stress. Notably, four hours after exposure to salt, significant activity was observed in the ABA, JA, IAA, and CTK pathways, with ABA, JA, JA-L-Ile, and IAA identified as key mediators in the response of Elymus sibiricus' to salinity. Moreover, SAPK3, Os04g0167900-like, CAT1, MKK2, and MPK12 were identified as potential central regulators within these pathways. The complex interactions between phytohormones and DEGs are crucial for facilitating the adaptation of Elymus sibiricus to saline environments. These findings enhance our understanding of the salt tolerance mechanisms in Elymus sibiricus and provide a foundation for breeding salt-resistant varieties.

PKCδ Protects against Lupus Autoimmunity.

Protein kinase C delta (PKCδ) has emerged as a key protective molecule against systemic lupus erythematosus (SLE or lupus), an autoimmune disease characterized by anti-double stranded (ds) DNA IgGs. Although PKCδ-deficient mice and lupus patients with mutated PRKCD genes clearly demonstrate the requirement for PKCδ in preventing lupus autoimmunity, this critical tolerance mechanism remains poorly understood. We recently reported that PKCδ acts as a key regulator of B cell tolerance by selectively deleting anti-dsDNA B cells in the germinal center (GC). PKCδ's tolerance function is activated by sphingomyelin synthase 2 (SMS2), a lipid enzyme whose expression is generally reduced in B cells from lupus patients. Moreover, pharmacologic strengthening of the SMS2/PKCδ tolerance pathway alleviated lupus pathogenesis in mice. Here, we review relevant publications in order to provide mechanistic insights into PKCδ's tolerance activity and discuss the potential significance of therapeutically targeting PKCδ's tolerance activity in the GC for selectively inhibiting lupus autoimmunity.

Abstract B003: Identification of novel genes that regulate aneuploidy tolerance by attenuating aneuploidy-induced stresses

Abstract Aneuploidy, an imbalanced number of chromosomes or chromosome arms, is the most common genetic aberration in cancer and is associated with a worse prognosis and advanced disease. However, in non-transformed cells, aneuploidy has a substantial fitness cost, leading to cell cycle arrest and cell death. This fitness cost is the result of several cellular stresses associated with aneuploidy, including proteotoxic, metabolic, mitotic and replication stress. Therefore, for aneuploidy to become beneficial, tolerance mechanisms that enable cancer cells to cope with aneuploidy-induced cellular stresses must come into play. Such mechanisms may uncover novel synthetic lethalities of aneuploid cancer cells. To this end, we systematically dissected the cellular mechanisms that facilitate aneuploidy tolerance. We combined large-scale analyses of gene expression, as well as genetic and pharmacological dependency datasets from human tumors, cancer cell lines and non-transformed cells, to identify genes and pathways associated with aneuploidy tolerance. We identified 55 genes associated (with high confidence) with aneuploidy tolerance. Gene set enrichment analysis (GSEA) revealed an enrichment for ribosome biogenesis and translation, DNA repair, replication stress and the p53 pathway. Interestingly, these pathways were all previously implicated with the aforementioned aneuploidy-induced cellular stresses. We plan to conduct a dependency mini-screen to validate the top ‘hits’ from our comprehensive bioinformatic analysis, and to explore the role of validated candidates in conferring aneuploidy tolerance. One of the top ‘hits’ of our exploratory analysis was a novel gene with an unclear cellular function. We experimentally confirmed that this gene was over-expressed in aneuploid cells, across several isogenic systems of matched diploid-aneuploid cell lines. Aneuploidy induction using MPS1 inhibitors led to its significant upregulation, demonstrating that its increased expression is a general feature of aneuploid cells. Mechanistically, we found that the novel gene had a role in regulating chromosome segregation, as its knockdown increased the rate of mitotic aberrations – as well as the sensitivity to mitotic checkpoint inhibition – resulting in increased chromosomal instability. We now study the molecular basis for the effect of this novel gene on aneuploidy tolerance. Specifically, we knocked it down in aneuploid cells, and over-expressed it in diploid cells, and we currently explore how these manipulations affect the above-mentioned aneuploidy tolerance pathways, namely replication stress, ribosome biogenesis, DNA damage repair and tp53 activation. In summary, we revealed a novel gene that is a key regulator of aneuploidy tolerance in cancer cells, likely due to its direct involvement in pathways required for the cellular coping with aneuploidy-induced stresses. Citation Format: Yonatan Eliezer, Adi V Tarrab, Tal Ben-Yishay, Hajime Okada, Tom Winkler, Uri Ben-David. Identification of novel genes that regulate aneuploidy tolerance by attenuating aneuploidy-induced stresses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B003.

Replication protein A dynamically re-organizes on primer/template junctions to permit DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis.

DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, PCNA, carry out DNA synthesis during lagging strand replication, initiation of leading strand replication, and the major DNA damage repair and tolerance pathways. Pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process involving the major single strand DNA (ssDNA)-binding protein complex, RPA, the processivity sliding clamp loader, RFC, PCNAand pol δ. During this process, the interactions of RPA, RFCand pol δ with a P/T junction all significantly overlap. A burning issue that has yet to be resolved is how these overlapping interactions are accommodated during this process. To address this, we design and utilize novel, ensemble FRET assays that continuously monitor the interactions of RPA, RFC, PCNAand pol δ with DNA as pol δ holoenzymes are assembled and initiate DNA synthesis. Results from the present study reveal that RPA remains engaged with P/T junctions throughout this process and the RPA•DNA complexes dynamically re-organize to allow successive binding of RFC and pol δ. These results have broad implications as they highlight and distinguish the functional consequences of dynamic RPA•DNA interactions in RPA-dependent DNA metabolic processes.

Response mechanism of Cynodon dactylon to flooding stress based on integrating metabonomics and transcriptomics analysis

Cynodon dactylon, a perennial herbaceous of Poaceae, possessing unique abilities in flooding tolerance and clonal propagation. However, evidences on molecular response mechanism from field are missing. Here, the differential responses of above-ground (stolon) and below-ground (root) organs of C. dactylon under flooding and non-flooding (CK) conditions were compared at the transcriptional and metabolomic levels. Under flooding stress, significant enrichment of differentially expressed genes (DEGs) related to dehydration responses, including cell water homeostasis and ion transmembrane transport were observed in both roots and stolons. The stolons were more sensitive to flooding, while enrichment in energy metabolism in roots were higher, such as the glycolytic process and glycogen biosynthetic process. The DEGs of photosynthesis in stolons were significantly down-regulated compared to roots. Under flooding stress, oxidative phosphorylation (OXPHOS) is inactivated, and the terminal oxidase of the mitochondrial electron transport chain (mtETC), cytochrome c oxidase (COX), is inhibited. Consequently, the expression of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) involved in fermentative metabolism is upregulated. The combined analysis indicated that phenylalanine, tyrosine, and tryptophan biosynthetic pathways in roots were significantly enriched in the flooding group. In contrast, there was a significant increase in phenylalanine biosynthesis in stolons, and the metabolite phenol exhibited up-regulated expression. In summary, phenylalanine metabolism and phenylalanine biosynthesis are the major responses to flooding stress, indicating the crucial role of these pathways in the flood tolerance of C. dactylon. These findings provide valuable evidence for understanding plant regulatory mechanisms underlying flooding tolerance.

Metabolomics and physio-chemical analyses of mulberry plants leaves response to manganese deficiency and toxicity reveal key metabolites and their pathways in manganese tolerance.

Manganese (Mn) plays a pivotal role in plant growth and development. Aside aiding in plant growth and development, Mn as heavy metal (HM) can be toxic in soil when applied in excess. Morus alba is an economically significant plant, capable of adapting to a range of environmental conditions and possessing the potential for phytoremediation of contaminated soil by HMs. The mechanism by which M. alba tolerates Mn stresses remains obscure. In this study, Mn concentrations comprising sufficiency (0.15 mM), higher regimes (1.5 mM and 3 mM), and deficiency (0 mM and 0.03 mM), were applied to M. alba in pot treatment for 21 days to understand M. alba Mn tolerance. Mn stress effects on the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), intercellular CO2 concentration (Ci), chlorophyll content, plant morphological traits, enzymatic and non-enzymatic parameters were analyzed as well as metabolome signatures via non-targeted LC-MS technique. Mn deficiency and toxicity decrease plant biomass, Pn, Ci, Gs, Tr, and chlorophyll content. Mn stresses induced a decline in the activities of catalase (CAT) and superoxide dismutase (SOD), while peroxidase (POD) activity, and leaf Mn content, increased. Soluble sugars, soluble proteins, malondialdehyde (MDA) and proline exhibited an elevation in Mn deficiency and toxicity concentrations. Metabolomic analysis indicates that Mn concentrations induced 1031 differentially expressed metabolites (DEMs), particularly amino acids, lipids, carbohydrates, benzene and derivatives and secondary metabolites. The DEMs are significantly enriched in alpha-linolenic acid metabolism, biosynthesis of unsaturated fatty acids, galactose metabolism, pantothenate and CoA biosynthesis, pentose phosphate pathway, carbon metabolism, etc. The upregulation of Galactinol, Myo-inositol, Jasmonic acid, L-aspartic acid, Coproporphyrin I, Trigonelline, Pantothenol, and Pantothenate and their significance in the metabolic pathways makes them Mn stress tolerance metabolites in M. alba. Our findings reveal the fundamental understanding of DEMs in M. alba's response to Mn nutrition and the metabolic mechanisms involved, which may hold potential significance for the advancement of M. alba genetic improvement initiatives and phytoremediation programs.

Unveiling immune tolerance pathways in preeclampsia placenta: implications for molecular targets and discovery of potential biomarkers.

During pregnancy, there is a link between disruption of maternal immune tolerance and preeclampsia, but the molecular mechanisms that regulate maternal and fetal immune tolerance remain unclear. This study employs bioinformatics to identify new markers related to placental immune tolerance and explore their potential role in predicting preeclampsia. Analyzing preeclampsia-related gene expression profiles in the Gene Expression Omnibus (GEO) dataset reveals 211 differentially expressed genes (DEGs) in the placenta, mainly influencing immune cell differentiation and response pathways. Employing weighted gene co-expression network analysis (WGCNA) and lasso regression, four potential target genes (ANKRD37, CRH, LEP, SIGLEC6) are identified for potential prediction of preeclampsia. Validation using the GSE4707 dataset confirmed the diagnostic and predictive potential of these candidate genes. RT-qPCR verified up-regulation in the placenta, while ELISA showed their correlation with immune tolerance factors associated with placental immune tolerance. As a result of this study, identifies potential biomarkers associated with placental immunity and contributes to understanding the molecular mechanism of preeclampsia.

PGPg_finder: A comprehensive and user-friendly pipeline for identifying plant growth-promoting genes in genomic and metagenomic data

Identifying and comparing plant growth-promoting traits (PGPT) within whole-genome and metagenomic sequencing data can significantly advance agricultural research and promote sustainable crop production. This study introduces PGPg_finder, a comprehensive pipeline designed to annotate and compare PGPT from both whole-genome and metagenome sequencing datasets. This pipeline utilizes direct sequence annotation alongside de novo assembly methods to accurately detect PGPT. By cross-referencing sequences from the PLaBAse database, it identifies and quantifies the presence of these genes within the original datasets, facilitating an intuitive comparison of the abundance and distribution of PGPT across various samples. We evaluated the performance of PGPg_finder by analyzing genomes from five rhizobacterial strains: Paenibacillus vini, Paenibacillus polymyxa, Fictibacillus sp., Brevibacillus agri, and Bacillus cereus, and also metagenomic samples from bulk soils subjected to forest-to-pasture conversion in the Amazon rainforest. The genomic workflow revealed several genes associated with substrate utilization, abiotic stress neutralization, phosphate solubilization, and iron acquisition. It also identified genes unique to specific lineages, including those associated with colonization and plant-derived substrate usage in P. polymyxa, quorum sensing response and biofilm formation in P. vini, heavy metal detoxification and nitrogen acquisition in B. agri, and spore production and neutralizing biotic stress in B. cereus. The strain Fictibacillus sp. presented several unique genes related to surface attachment, stress response, xenobiotic degradation, phosphate solubilization, and phytohormone production. The use of PGPg_finder highlights its potential to uncover novel inoculants and strains. The metagenomic workflow distinguished plant-growth promotion gene profiles between soils from the Amazon rainforest and pasture, with the latter showing a profile more aligned with simple carbohydrate consumption, abiotic stress tolerance, motility and chemotaxis, and phosphorus mineralization. Native forests exhibited a profile associated with the degradation of complex organic matter, oxidative stress tolerance, xenobiotic degradation, bactericidal activity, iron acquisition, and volatile pathways. These findings underscore the effectiveness and sensitivity of PGPg_finder in accurately identifying and comparing PGPT genes, highlighting both commonalities and variations across samples. The application of this pipeline has the potential to significantly facilitate the identification of plant growth-promoting microbes.

Molecular Mechanisms of CBL-CIPK Signaling Pathway in Plant Abiotic Stress Tolerance and Hormone Crosstalk.

Abiotic stressors, including drought, salt, cold, and heat, profoundly impact plant growth and development, forcing elaborate cellular responses for adaptation and resilience. Among the crucial orchestrators of these responses is the CBL-CIPK pathway, comprising calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs). While CIPKs act as serine/threonine protein kinases, transmitting calcium signals, CBLs function as calcium sensors, influencing the plant's response to abiotic stress. This review explores the intricate interactions between the CBL-CIPK pathway and plant hormones such as ABA, auxin, ethylene, and jasmonic acid (JA). It highlights their role in fine-tuning stress responses for optimal survival and acclimatization. Building on previous studies that demonstrated the enhanced stress tolerance achieved by upregulating CBL and CIPK genes, we explore the regulatory mechanisms involving post-translational modifications and protein-protein interactions. Despite significant contributions from prior research, gaps persist in understanding the nuanced interplay between the CBL-CIPK system and plant hormone signaling under diverse abiotic stress conditions. In contrast to broader perspectives, our review focuses on the interaction of the pathway with crucial plant hormones and its implications for genetic engineering interventions to enhance crop stress resilience. This specialized perspective aims to contribute novel insights to advance our understanding of the potential of the CBL-CIPK pathway to mitigate crops' abiotic stress.

E3 ubiquitin ligase RNF2 protects polymerase ι from destabilization

Human DNA polymerase ι (Polι) belongs to the Y-family of specialized DNA polymerases engaged in the DNA damage tolerance pathway of translesion DNA synthesis that is crucial to the maintenance of genome integrity. The extreme infidelity of Polι and the fact that both its up- and down-regulation correlate with various cancers indicate that Polι expression and access to the replication fork should be strictly controlled. Here, we identify RNF2, an E3 ubiquitin ligase, as a new interacting partner of Polι that is responsible for Polι stabilization in vivo. Interestingly, while we report that RNF2 does not directly ubiquitinate Polι, inhibition of the E3 ubiquitin ligase activity of RNF2 affects the cellular level of Polι thereby protecting it from destabilization. Additionally, we indicate that this mechanism is more general, as DNA polymerase η, another Y-family polymerase and the closest paralogue of Polι, share similar features.

Interactive abiotic and biotic stressor impacts on a stream-dwelling amphibian.

Organisms within freshwater and marine environments are subject to a diverse range of often co-occurring abiotic and biotic stressors. Despite growing awareness of the complex multistress systems at play in aquatic ecosystems, many questions remain regarding how simultaneous stressors interact with one another and jointly impact aquatic species. We looked at multistress interactions in a protected stream ecosystem in Mendocino County, California. Specifically, we examined how diurnal temperature variation, turbidity, and predator cues altered the movement speed of larval Pacific giant salamanders (Dicamptodon tenebrosus). In a second experiment, we looked at how simulated low-flow summer conditions impact the expression of heat-shock proteins (HSPs) in the same species. Larvae moved almost one and a half times faster in the presence of chemical cues from trout and suspended sediment, and almost two times faster when both sediment and trout cues were present but were only marginally affected by temperature and visual cues from conspecifics. Interestingly, the order of stressor exposure also appeared to influence larval speed, where exposure to sediment and trout in earlier trials tended to lead to faster speeds in later trials. Additionally, larvae exposed to low-flow conditions had more variable, but not statistically significantly higher, expression of HSPs. Our findings highlight the potential interactive effects of an abiotic stressor, sedimentation, and a biotic stressor, and predator chemical cues on an ecologically important trait: movement speed. Our findings also demonstrate the likely role of HSPs in larval salamander survival in challenging summer conditions. Taken together, these findings show that larval D. tenebrosus responds behaviorally to biotic and abiotic stressors and suggests a possible pathway for physiological tolerance of environmental stress. Consideration of multistress systems and their effects is important for understanding the full effects of co-occurring stressors on aquatic organisms to guide appropriate conservation and management efforts based on ecologically relevant responses of organisms within an environment.

Mitigating Effect of Trans-Zeatin on Cadmium Toxicity in Desmodesmus armatus.

Phytohormones, particularly cytokinin trans-zeatin (tZ), were studied for their impact on the green alga Desmodesmus armatus under cadmium (Cd) stress, focusing on growth, metal accumulation, and stress response mechanisms. Using atomic absorption spectroscopy for the Cd level and high-performance liquid chromatography for photosynthetic pigments and phytochelatins, along with spectrophotometry for antioxidants and liquid chromatography-mass spectrometry for phytohormones, we found that tZ enhances Cd uptake in D. armatus, potentially improving phycoremediation of aquatic environments. Cytokinin mitigates Cd toxicity by regulating internal phytohormone levels and activating metal tolerance pathways, increasing phytochelatin synthase activity and phytochelatin accumulation essential for Cd sequestration. Treatment with tZ and Cd also resulted in increased cell proliferation, photosynthetic pigment and antioxidant levels, and antioxidant enzyme activities, reducing oxidative stress. This suggests that cytokinin-mediated mechanisms in D. armatus enhance its capacity for Cd uptake and tolerance, offering promising avenues for more effective aquatic phycoremediation techniques.

A Stem-like Patient-Derived Ovarian Cancer Model of Platinum Resistance Reveals Dissociation of Stemness and Resistance.

To understand chemoresistance in the context of cancer stem cells (CSC), a cisplatin resistance model was developed using a high-grade serous ovarian cancer patient-derived, cisplatin-sensitive sample, PDX4. As a molecular subtype-specific stem-like cell line, PDX4 was selected for its representative features, including its histopathological and BRCA2 mutation status, and exposed to cisplatin in vitro. In the cisplatin-resistant cells, transcriptomics were carried out, and cell morphology, protein expression, and functional status were characterized. Additionally, potential signaling pathways involved in cisplatin resistance were explored. Our findings reveal the presence of distinct molecular signatures and phenotypic changes in cisplatin-resistant PDX4 compared to their sensitive counterparts. Surprisingly, we observed that chemoresistance was not inherently linked with increased stemness. In fact, although resistant cells expressed a combination of EMT and stemness markers, functional assays revealed that they were less proliferative, migratory, and clonogenic-features indicative of an underlying complex mechanism for cell survival. Furthermore, DNA damage tolerance and cellular stress management pathways were enriched. This novel, syngeneic model provides a valuable platform for investigating the underlying mechanisms of cisplatin resistance in a clinically relevant context, contributing to the development of targeted therapies tailored to combat resistance in stem-like ovarian cancer.

Role of Rad18 in B cell activation and lymphomagenesis

Maintenance of genome integrity is instrumental in preventing cancer. In addition to DNA repair pathways that prevent damage to DNA, damage tolerance pathways allow for the survival of cells that encounter DNA damage during replication. The Rad6/18 pathway is instrumental in this process, mediating damage bypass by ubiquitination of proliferating cell nuclear antigen. Previous studies have shown different roles of Rad18 in vivo and in tumorigenesis. Here, we show that B cells induce Rad18 expression upon proliferation induction. We have therefore analysed the role of Rad18 in B cell activation as well as in B cell lymphomagenesis mediated by an Eµ–Myc transgene. We find no activation defects or survival differences between Rad18 WT mice and two different models of Rad18 deficient tumour mice. Also, tumour subtypes do not differ between the mouse models. Accordingly, functions of Rad18 in B cell activation and tumorigenesis may be compensated for by other pathways in B cells.

Abstract 864: Pan-cancer analysis of lactic acid metabolism landscape across the Cancer Genome Atlas

Abstract Introduction: Metabolic reprogramming plays an important role in tumor occurrence and progression. Our studies confirmed the survival state of patients with different metabolic systems was heterogeneous and glycolytic metabolism may cause tumor progression. Based on these findings, lactic acid metabolism in glycolysis aroused our interest. This study evaluated the potential association between lactate metabolism gene and tumor clinical process in hepatocellular carcinoma, and proved that it can be used as an effective tool for survival prediction and treatment guidance of 32 kinds of tumor patients by establishing lactate score prognosis model. Methods: We used the expression profile, proteomics and epigenetics of HCC lactate gene set in the Cancer Genome Atlas Database to systematically describe the changes of lactate related genes. The differentially expressed genes were then verified in 32 clinical tumor data sets. A prognostic model of tumor lactic acid infiltration was constructed to evaluate its correlation with the prognosis of patients. Finally, we analyzed the effects of lame score, immunotherapy and drug response on the clinical process of tumor. Results: Firstly, we analyzed the differential expression of lactate metabolism genes in HCC and found that the prognosis of patients with high lactate infiltration was significantly lower than that of mild infiltration. AF angiogenesis factor and EMT factor in high infiltration may be the main reason for their differential expression. We constructed a tumor lactic acid prognosis model through six significantly different lactic acid genes, and found that lame score can be used as a predictor of immunotherapy. In addition, we proved the role of lame score in Pan cancer. PGA was positively correlated with lame score in most tumors. The prognosis of 16 tumors with high lame score group was significantly lower than that of low lame score group. There were significant differences in lame score between metastatic and non metastatic cancer groups of Cesc, kirc and kirp. The high and low risk groups of some tumors showed different dryness indices and dampness. At the same time, methylation gene modification has a significant impact on the Lame score of pan cancer, and the Lame score is used to link the drug tolerance and specific pathway enrichment of pan cancer tumor cell lines, confirming the targeted effect of Lame score on tumor treatment. Finally, we used the clinical characteristics and immunohistochemical analysis of a cohort study of real patients with breast cancer endocrine therapy (Tamoxifen) and hepatocellular carcinoma targeted therapy (Lenvartinib) to verify the clinical treatment prediction ability of lame score. Conclusions: Our study provides a comprehensive analysis of lactate metabolic across cancers and highlights the potential treatment of lactate targeted therapies for cancers. Citation Format: Xiaolin Wei, Lei Cai, Qian He. Pan-cancer analysis of lactic acid metabolism landscape across the Cancer Genome Atlas [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 864.

Meta-analysis of public RNA sequencing data of abscisic acid-related abiotic stresses in Arabidopsis thaliana.

Abiotic stresses such as drought, salinity, and cold negatively affect plant growth and crop productivity. Understanding the molecular mechanisms underlying plant responses to these stressors is essential for stress tolerance in crops. The plant hormone abscisic acid (ABA) is significantly increased upon abiotic stressors, inducing physiological responses to adapt to stress and regulate gene expression. Although many studies have examined the components of established stress signaling pathways, few have explored other unknown elements. This study aimed to identify novel stress-responsive genes in plants by performing a meta-analysis of public RNA sequencing (RNA-Seq) data in Arabidopsis thaliana, focusing on five ABA-related stress conditions (ABA, Salt, Dehydration, Osmotic, and Cold). The meta-analysis of 216 paired datasets from five stress conditions was conducted, and differentially expressed genes were identified by introducing a new metric, called TN [stress-treated (T) and non-treated (N)] score. We revealed that 14 genes were commonly upregulated and 8 genes were commonly downregulated across all five treatments, including some that were not previously associated with these stress responses. On the other hand, some genes regulated by salt, dehydration, and osmotic treatments were not regulated by exogenous ABA or cold stress, suggesting that they may be involved in the plant response to dehydration independent of ABA. Our meta-analysis revealed a list of candidate genes with unknown molecular mechanisms in ABA-dependent and ABA-independent stress responses. These genes could be valuable resources for selecting genome editing targets and potentially contribute to the discovery of novel stress tolerance mechanisms and pathways in plants.

Abstract 670: Preclinical candidate LAE120, a novel selective USP1 inhibitor shows effective anticancer and combination activity with PARP inhibitor

Abstract The Fanconi anemia (FA) and translesion synthesis (TLS) are DNA damage tolerance and repair pathways that have been found to be regulated by reversible ubiquitination in which ubiquitin-specific protease 1 (USP1) plays a key role. Numerous studies have indicated that USP1 inhibitors have therapeutic effects on tumors with homologous recombination deficiency (HRD), including mutations in BRCA1/2. Due to the emergence of resistance to PARP inhibitors in clinical applications, new strategies are needed to address this issue. Here we present LAE120, a novel, allosteric and highly potent USP1 inhibitor, displayed monotherapy potency and combination activity with PARP inhibitor in HRD cancers. LAE120 has a unique tail group distinct from all the published USP1 inhibitors and is predicted to induce a different conformational change at the USP1 allosteric site. In the biochemical assay, LAE120 strongly inhibited USP1 enzymatic activity with IC50 of 5.6 nM. LAE120 potently inhibited proliferation of BRCA-mutated MDA-MB-436 breast cancer cell (IC50:19 nM) and exhibited anti-proliferative activity in NCI-H1792 (IC50: 69 nM) and K562 (IC50: 38 nM) cell lines. In the xenograft model of MDA-MB-436 cells with BRCA mutations, LAE120 demonstrated robust single-agent tumor inhibitory activity (TGI: 88%, 10 mg/kg BID) and a favorable dose-dependent response (TGI: 106%, 25 mg/kg BID). We also observed strong tumor-inhibition efficacy of LAE120 as a single agent in the K562 xenograft model with Bcr-Abl expression (TGI: 86%, 10 mg/kg BID). Current research indicates that USP1 inhibitors exhibit synergistic activity in combination with PARP inhibitors and re-sensitizes PARP refractory tumors. Similar results have been confirmed in the xenograft model of BRCA-mutated MDA-MB-436 cells. The combination of LAE120 and Olaparib (PARP inhibitor) yielded a more robust and durable anti-tumor efficacy at low dose (TGI: 110%, LAE120, 5 mg/kg BID + Olaparib, 50 mg/kg QD). LAE120 also displayed favorable ADME properties and PK profiles. Preclinical-tox studies indicated the molecule was well tolerated without significant toxicities and hematological observations. Both in vitro and in vivo data strongly support further investigation of LAE120, whether as a single agent or in combination therapy. Citation Format: Jintao Wang, Yan Chen, Junyan Chen, Ling Jiang, Xiaofen Lin, Chaojun Cai, Minhua Zhang, Ming Li, Justin Gu. Preclinical candidate LAE120, a novel selective USP1 inhibitor shows effective anticancer and combination activity with PARP inhibitor [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 670.

Abstract 4527: TNG348 is synergistic with PARP inhibitors in tumor models with elevated replication stress

Abstract Defective maintenance of genomic integrity is a hallmark of cancer cells that can result from oncogene-induced replication stress and by loss of DNA repair mechanisms. DNA repair deficiencies and elevated replication stress present targetable vulnerabilities for cancer treatment. Notably, BRCA1/2 mutant and homologous recombination deficient (HRD) tumors cannot repair double-strand breaks by homologous recombination and rely on alternative pathways of DNA repair. PARP inhibitors (PARPi), which are a standard of care in many BRCA1/2 mutant tumors, cause synthetic lethality with BRCA1/2 mutation by inhibiting the DNA base excision repair pathway. Despite the clinical benefit of PARPi, they are not effective in every HRD tumor and the acquisition of PARPi resistance limits long-term response. TNG348, a selective allosteric inhibitor of the deubiquitinating enzyme USP1, was specifically designed to target HRD vulnerabilities through an alternative mechanism. We previously showed that the anti-tumor activity of USP1 inhibition results from disruption of the translesion synthesis DNA damage tolerance pathway, a mechanism of action that is functionally distinct from base excision repair targeted by PARPi. Our preclinical studies show that TNG348 is active in HRD models and strongly synergizes with PARP inhibitors to drive strong anti-tumor responses. We have identified replication stress as a predictive biomarker of TNG348 response using cell line profiling and genome-wide CRISPR screens. For example, overexpression of oncogenes known to induce replication stress sensitized to the TNG348 and PARPi combination both in vitro and in vivo. These data indicate that cancer-specific elevated DNA replication stress could contribute to tumor sensitivity to TNG348 and provide additional patient stratification strategies and opportunities for indication expansion. Citation Format: Antoine Simoneau, Hsin-Jung Wu, Charlotte Pratt, Grace Comer, Shangtao Liu, Samuel Meier, Tenzing Khendu, Ashley Choi, Hongxiang Zhang, Binzhang Shen, Douglas Whittington, Sirimas Sudsakorn, Wenhai Zhang, Yi Yu, Yingnan Chen, Brian Haines, Adam Crystal, Jannik N. Andersen, John Maxwell, Scott Throner. TNG348 is synergistic with PARP inhibitors in tumor models with elevated replication stress [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 4527.

Dominant roles of BRCA1 in cellular tolerance to a chain-terminating nucleoside analog, alovudine

Alovudine is a chain-terminating nucleoside analog (CTNA) that is frequently used as an antiviral and anticancer agent. Generally, CTNAs inhibit DNA replication after their incorporation into nascent DNA during DNA synthesis by suppressing subsequent polymerization, which restricts the proliferation of viruses and cancer cells. Alovudine is a thymidine analog used as an antiviral drug. However, the mechanisms underlying the removal of alovudine and DNA damage tolerance pathways involved in cellular resistance to alovudine remain unclear. Here, we explored the DNA damage tolerance pathways responsible for cellular tolerance to alovudine and found that BRCA1-deficient cells exhibited the highest sensitivity to alovudine. Moreover, alovudine interfered with DNA replication in two distinct mechanisms: first: alovudine incorporated at the end of nascent DNA interfered with subsequent DNA synthesis; second: DNA replication stalled on the alovudine-incorporated template strand. Additionally, BRCA1 facilitated the removal of the incorporated alovudine from nascent DNA, and BRCA1-mediated homologous recombination (HR) contributed to the progressive replication on the alovudine-incorporated template. Thus, we have elucidated the previously unappreciated mechanism of alovudine-mediated inhibition of DNA replication and the role of BRCA1 in cellular tolerance to alovudine.

Inhibition of spinal BRD4 alleviates pyroptosis and M1 microglia polarization via STING-IRF3 pathway in morphine-tolerant rats

BackgroundMorphine tolerance has been a challenging medical issue. Neuroinflammation is considered as a critical mechanism for the development of morphine tolerance. Bromodomain-containing protein 4 (BRD4), a key regulator in cell damage and inflammation, participates in the development of chronic pain. However, whether BRD4 is involved in morphine tolerance and the underlying mechanisms remain unknown. MethodsThe morphine-tolerant rat model was established by intrathecal administration of morphine twice daily for 7 days. Behavior test was assessed by a tail-flick latency test. The roles of BRD4, pyroptosis, microglia polarization and related signaling pathways in morphine tolerance were elucidated by Western blot, real-time quantitative polymerase chain reaction, and immunofluorescence. ResultsRepeated morphine administration upregulated BRD4 level, induced pyroptosis, and promoted microglia M1-polarization in spinal cord, accompanied by the release of proinflammatory cytokines, such as TNF-α and IL-1β. JQ-1, a BRD4 antagonist, alleviated the development of morphine tolerance, diminished pyroptosis and induced the switch of microglia from M1 to M2 phenotype. Mechanistically, stimulator of interferon gene (STING)- interferon regulatory factor 3 (IRF3) pathway was activated and the protective effect of JQ-1 against morphine tolerance was at least partially mediated by inhibition of STING-IRF3 pathway. ConclusionThis study demonstrated for the first time that spinal BRD4 contributes to pyroptosis and switch of microglia polarization via STING-IRF3 signaling pathway during the development of morphine tolerance, which extend the understanding of the neuroinflammation mechanism of morphine tolerance and provide an alternative strategy for the precaution against of this medical condition.