Wild-type Cells Research Articles

Inhibition of P21-activated kinases 1 and 4 synergistically suppresses the growth of pancreatic cancer by stimulating anti-tumour immunity

BackgroundPancreatic ductal adenocarcinoma (PDA) is one of the most lethal types of cancer, and KRAS oncogene occurs in over 90% of cases. P21-activated kinases (PAK), containing six members (PAK1 to 6), function downstream of KRAS. PAK1 and PAK4 play important roles in carcinogenesis, but their combinational effect remains unknown. In this study, we have determined the effect of dual inhibition of PAK1 and PAK4 in PDA progression using knockout (KO) cancer cell lines.MethodsMurine wild-type (WT) and PAK1KO pancreatic cancer cell lines were isolated from PAK1+/+ and PAK1−/− KPC (LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx-1-Cre) mice. KPC PAK4KO and KPC PAK1&4 KO cell lines were generated from KPC WT and KPC PAK1KO cell lines respectively using the CRISPR-CAS9 gene knockout technique. PAK WT and KO cell lines were used in mouse models of pancreatic tumours. Cells and tumour tissue were also used in flow cytometry and proteomic studies. A human PDA tissue microarray was stained by immunohistochemistry.ResultsDouble knock out of PAK1 and PAK4 caused complete regression of tumour in a syngeneic mouse model. PAK4KO inhibited tumour growth by stimulating a rapid increase of cytotoxic CD8+ T cell infiltration. PAK1KO synergistically with PAK4KO increased cytotoxic CD8+ T cell infiltration and stimulated a sustained infiltration of CD8+ T cells at a later phase to overcome the immune evasion in the PAK4KO tumour. The human PDA tissue microarray study showed the important role of PAK1 and PAK4 in intra-tumoral T-cell function.ConclusionOur results demonstrated that dual inhibition of PAK1 and PAK4 synergistically suppressed PDA progression by stimulating cytotoxic CD8 + T cell response.

Macrophage scavenger receptor-A1 promotes skeletal muscle regeneration after hindlimb ischemia.

Macrophages mediated inflammatory response is crucial for the recovery of skeletal muscle following ischemia. Thus, it's necessary to exploit macrophages based therapeutic targets for ischemic disease. Here, we found mRNA level of SR-A1 was elevated in patients with critical limb ischemia by analysis of gene expression omnibus (GEO) database. Then we investigated the role and the underlined mechanisms of macrophage SR-A1 in a mouse HLI model. Compared with the SR-A1 fl/fl mice, the Lyz Cre/+/SR-A1 flox/flox (SR-A1 ΔMΦ) mice showed significantly lower laser doppler blood flow in the ischemic limb at day 7 after HLI. Consistently, histological analysis exhibited that ischemic limb of SR-A1 ΔMΦ mice displayed more sever and sustained necrotic morphology, inflammation and fibrosis, decreased vessel density and regeneration rate, compared with which of control SR-A1 fl/fl mice. Furthermore, restoration of wild-type myeloid cells to SR-A1 knock-out mice effectively relieved the doppler perfusion in the ischemic limb and restrained skeletal muscle damage 7 days post HLI. In line with in vivo findings, when co-cultivating macrophages with the mouse myoblast line C2C12, SR-A1 -/- bone marrow macrophage significantly inhibited myoblast differentiation in vitro. Mechanically, SR-A1 enhanced skeletal muscle regeneration response to HLI by inhibiting the oncostatin M (OSM) production via suppressed NF-κB signaling activation. These results indicates that SR-A1 is a promising candidate molecule to improve tissue repair and regeneration in peripheral ischemic arterial disease.

#1236 Translation reprogramming in autosomal dominant polycystic kidney disease

Abstract Background and Aims Global regulation of mRNA translation has emerged as a central mechanism driving cellular adaptation to innate or acquired metabolic challenges. tRNA modifications are emerging as key drivers in the reprogramming of mRNA translation. Metabolic dysregulation is a central feature in the pathophysiology of PKD1-associated autosomal dominant polycystic kidney disease (ADPKD). Here, we aim at investigating the putative role of mRNA translation regulation in the development of cysts in ADPKD. Method The classical murine Inner Medullary Collecting Duct (IMCD) cells knocked out for Pkd1 by CRISPR-Cas9 were used. We combined proteomics and transcriptomics analyses of wild-type versus Pkd1-KO IMCD cells. A series of bioinformatic tools was used to identify pathways and processes dysregulated at the level of mRNA translation. Moreover, global mRNA translation rates were assessed in Pkd1-KO cells by measuring translating ribosomes and nascent protein synthesis by flow cytometry labelling methods. Finally, systematic analyses of tRNA regulation (sequencing and modification) were performed in Pkd1-KO cells. Results The proposed combination of analyses highlighted pathways differentially regulated in Pkd1-KO cells. We focused on pathways and proteins significantly upregulated in Pkd1-KO as compared to controls cells. On top of expected up-regulated pathways, we also uncovered a series of specific cellular and molecular pathways that are specifically and significantly upregulated in Pkd1-KO cells, including intracellular vesicular metabolism and ciliary transport. Interestingly, the pathway “tRNA modification” was significantly enriched in Pkd1-KO cells. Given the role of tRNA modifications in translation reprogramming during cell adaptation, we further validated that several enzymes known to regulate tRNA modification were differentially expressed in Pkd1-KO cells. In order to investigate the changes in tRNA modifications taking place in Pkd1-KO cells, we performed an unbiased analysis of tRNA modifications (LC-MS). This uncovered reproducible differences in the abundance of specific tRNA modifications between control and Pkd1-KO IMCD cells. Conclusion Our data uncovered different patterns of tRNA modifications between Pkd1-KO versus wild-type IMCD cells. This suggests that a specific tRNA-modification-dependent reprogramming of mRNA translation may occur in ADPKD. These data will be further validated using patient derived ADPKD cyst models.

#2695 A Polycystin-1-Bicaudal C1 complex regulates focal adhesion function through integrin beta-1, F-actin organisation and local translation in HKE cells

Abstract Background and Aims Variants in Bicc1, result in a recessive murine PKD phenotype (bpk, jcpk) by uncertain mechanisms. In a recent study, we reported genetic and biochemical evidence of novel interactions between PKD1, PKD2 and BICC1 in Xenopus, mouse and man suggesting that BICC1 is a modifier of the ADPKD phenotype (submitted). In this study, our major aim was to investigate the molecular mechanisms underlying cystogenesis that result from loss of the normal interaction between the PKD1 protein, Polycystin-1 (PC1), a cell surface receptor and Bicaudal C1 (BICC1), an RNA-binding protein. Methods Isogenic PKD1 and BICC1 CRISP-Cas9 KO cells were generated from a conditionally-immortalised wild-type human proximal tubular line (UCL93); RNA-seq was performed in PKD1 and BICC1 KO cells to identify commonly altered genes and pathways; MS pull-down was conducted in HEK293 cells with BICC1-HA to identify BICC1 interacting proteins; cellular phenotypes in normal and KO cells were assessed through adhesion and migration assays, alongside Traction Force Microscopy (TFM); proximal ligation assays (PLA) to confirm protein-protein interactions; single-molecule FISH (smFISH) to localize mRNA transcripts; puromycin labeling with a proximity ligation assay (Puro-PLA) to detect sites of translation of specific mRNAs. Results Consistent with a functional interaction, loss of PC1 resulted in lower BICC1 levels and vice-versa in single KO (SKO) cells. In migrating cells, BICC1 was expressed at the leading edge where it co-localised with β1-integrin (ITGB1) by PLA, suggesting enrichment at focal adhesion sites (FA). BICC1 KO cells had no cilia phenotype but instead displayed striking disorganization of stress fibres and aster-like structures, features which could be completely rescued by lentiviral Bicc1-HA re-expression. Gene set enrichment and pathway analysis confirmed that F-actin organization and actin-associated proteins (AAP) were commonly dysregulated in SKO cells. BICC1 was also found to interact ITGB1, several components of an integrin-associated complex and proteins involved in actin turnover by MS pull-down. Both SKO cells showed major abnormalities of cell attachment, detachment and directional migration indicative of functional alterations in cell-matrix and cell-cell adhesion. We confirmed that FA structure and function were altered by reduced co-localisation of several AAPs (including TNS1 and FBLIM1) and by TFM. BICC1 directly interacted with F-actin and FBLIM1 in pull-down assays and with FBLIM1 by RNA-IP. Finally, we found evidence of FBLIM1 mRNA by smFISH and of local FBLIM1 translation (Puro-PLA) at FA sites in wild-type cells, both of which were markedly reduced in SKO cells. Conclusion Our data reveal the key roles for actin dynamics and local translation as novel mechanisms regulated by PC1 and BICC1 in the pathogenesis of ADPKD. We demonstrate that BICC1 and PC1 are co-regulated, forming a PC1-BICC1 complex critical for maintaining the structural integrity and function of FAs in normal kidney epithelial cells. Loss of this interaction results in major alterations in cell adhesion, migration and stress fibre organisation, features typical of the ADPKD cellular phenotype. BICC1 plays a dual role in this process via protein-protein interactions with ITGB1, F-actin and other AAPs, and through protein-RNA interactions regulating the local translation of key AAPs (FBLIM1) at FA sites.

#2490 Analyzing the morphology of mesothelial cells when exposed to peritoneal dialysis solutions

Abstract Background and Aims Glucose remains the predominant osmotic agent in peritoneal dialysis (PD), yet its prolonged use is associated with an inevitable decline in peritoneal filtration capacity. Persistent contact with glucose during dialysis can induce significant morphological and functional changes in the peritoneum. In this study, we explore the morphological alterations in mesothelial cells exposed to both conventional glucose-based PD solutions and an innovative biocompatible PD solution (XyloCore) containing L-carnitine and Xylitol. Method Met5A mesothelial cells were seeded and cultured on a polyester filter (with a pore size of 0.4 micrometers, Transwell, 12 well type, Millipore) utilizing a full medium. The impact of PD solutions, differentiating between glucose-based and XyloCore, was assessed on cells cultivated on transwells. These cells were exposed to PD solutions on the apical side and to culture medium on the basal side. The cells were then employed for proteomic and metabolomic assessments and were also fixed and scrutinized through scanning electron microscopy (SEM). Results Through unsupervised hierarchical clustering analysis, discernable proteomic distinctions emerged between wild-type (WT) cells (control) and cells exposed to either glucose-based PD solution or XyloCore. In SEM analysis, control cells exhibited a distinct flat epithelial appearance. Contrastingly, subjecting WT cells to treatment with a glucose-based PD solution induced significant alterations in cellular morphology. Notably, cells displayed elongated shapes, loosening of cell-cell contacts, and a consistent denuding of the culture surface. In contrast, XyloCore maintained a better cellular morphology and mitigated cell detachment. Conclusion Preserving the integrity of the mesothelial barrier is crucial for maintaining peritoneal ultrafiltration capacity. The findings suggest that glucose-based PD solutions trigger early morphological changes in cell shape and cell-cell contacts, likely stemming from alterations in cytoskeleton or adhesion molecules. The exploration of new osmotic agents in clinical practice holds paramount significance for the future of peritoneal dialysis.

Eliminating elevated p53 signaling fails to rescue skeletal muscle defects or extend survival in lamin A/C-deficient mice.

Lamins A and C, encoded by the LMNA gene, are nuclear intermediate filaments that provide structural support to the nucleus and contribute to chromatin organization and transcriptional regulation. LMNA mutations cause muscular dystrophies, dilated cardiomyopathy, and other diseases. The mechanisms by which many LMNA mutations result in muscle-specific diseases have remained elusive, presenting a major hurdle in the development of effective treatments. Previous studies using striated muscle laminopathy mouse models found that cytoskeletal forces acting on mechanically fragile Lmna-mutant nuclei led to transient nuclear envelope rupture, extensive DNA damage, and activation of DNA damage response (DDR) pathways in skeletal muscle cells in vitro and in vivo. Furthermore, hearts of Lmna mutant mice have elevated activation of the tumor suppressor protein p53, a central regulator of DDR signaling. We hypothesized that elevated p53 activation could present a pathogenic mechanism in striated muscle laminopathies, and that eliminating p53 activation could improve muscle function and survival in laminopathy mouse models. Supporting a pathogenic function of p53 activation in muscle, stabilization of p53 was sufficient to reduce contractility and viability in wild-type muscle cells in vitro. Using three laminopathy models, we found that increased p53 activity in Lmna-mutant muscle cells primarily resulted from mechanically induced damage to the myonuclei, and not from altered transcriptional regulation due to loss of lamin A/C expression. However, global deletion of p53 in a severe muscle laminopathy model did not reduce the disease phenotype or increase survival, indicating that additional drivers of disease must contribute to the disease pathogenesis.

ShRNA-mediated gene silencing of HDAC11 empowers CAR-T cells against prostate cancer

Epigenetic mechanisms are involved in several cellular functions, and their role in the immune system is of prime importance. Histone deacetylases (HDACs) are an important set of enzymes that regulate and catalyze the deacetylation process. HDACs have been proven beneficial targets for improving the efficacy of immunotherapies. HDAC11 is an enzyme involved in the negative regulation of T cell functions. Here, we investigated the potential of HDAC11 downregulation using RNA interference in CAR-T cells to improve immunotherapeutic outcomes against prostate cancer. We designed and tested four distinct short hairpin RNA (shRNA) sequences targeting HDAC11 to identify the most effective one for subsequent analyses. HDAC11-deficient CAR-T cells (shD-NKG2D-CAR-T) displayed better cytotoxicity than wild-type CAR-T cells against prostate cancer cell lines. This effect was attributed to enhanced activation, degranulation, and cytokine release ability of shD-NKG2D-CAR-T when co-cultured with prostate cancer cell lines. Our findings reveal that HDAC11 interference significantly enhances CAR-T cell proliferation, diminishes exhaustion markers PD-1 and TIM3, and promotes the formation of T central memory TCM populations. Further exploration into the underlying molecular mechanisms reveals increased expression of transcription factor Eomes, providing insight into the regulation of CAR-T cell differentiation. Finally, the shD-NKG2D-CAR-T cells provided efficient tumor control leading to improved survival of tumor-bearing mice in vivo as compared to their wild-type counterparts. The current study highlights the potential of HDAC11 downregulation in improving CAR-T cell therapy. The study will pave the way for further investigations focused on understanding and exploiting epigenetic mechanisms for immunotherapeutic outcomes.

Nε-lysine acetylation of the histone-like protein HBsu influences antibiotic survival and persistence in Bacillus subtilis

Nε-lysine acetylation is recognized as a prevalent post-translational modification (PTM) that regulates proteins across all three domains of life. In Bacillus subtilis, the histone-like protein HBsu is acetylated at seven sites, which regulates DNA compaction and the process of sporulation. In Mycobacteria, DNA compaction is a survival strategy in response antibiotic exposure. Acetylation of the HBsu ortholog HupB decondenses the chromosome to escape this drug-induced, non-growing state, and in addition, regulates the formation of drug-tolerant subpopulations by altering gene expression. We hypothesized that the acetylation of HBsu plays similar regulatory roles. First, we measured nucleoid area by fluorescence microscopy and in agreement, we found that wild-type cells compacted their nucleoids upon kanamycin exposure, but not exposure to tetracycline. We analyzed a collection of HBsu mutants that contain lysine substitutions that mimic the acetylated (glutamine) or unacetylated (arginine) forms of the protein. Our findings indicate that some level of acetylation is required at K3 for a proper response and K75 must be deacetylated. Next, we performed time-kill assays of wild-type and mutant strains in the presence of different antibiotics and found that interfering with HBsu acetylation led to faster killing rates. Finally, we examined the persistent subpopulation and found that altering the acetylation status of HBsu led to an increase in persister cell formation. In addition, we found that most of the deacetylation-mimic mutants, which have compacted nucleoids, were delayed in resuming growth following removal of the antibiotic, suggesting that acetylation is required to escape the persistent state. Together, this data adds an additional regulatory role for HBsu acetylation and further supports the existence of a histone-like code in bacteria.

Targeting host O-linked glycan biosynthesis affects Ebola virus replication efficiency and reveals differential GalNAc-T acceptor site preferences on the Ebola virus glycoprotein.

Ebola virus glycoprotein (EBOV GP) is one of the most heavily O-glycosylated viral glycoproteins, yet we still lack a fundamental understanding of the structure of its large O-glycosylated mucin-like domain and to what degree the host O-glycosylation capacity influences EBOV replication. Using tandem mass spectrometry, we identified 47 O-glycosites on EBOV GP and found similar glycosylation signatures on virus-like particle- and cell lysate-derived GP. Furthermore, we performed quantitative differential O-glycoproteomics on proteins produced in wild-type HEK293 cells and cell lines ablated for the three key initiators of O-linked glycosylation, GalNAc-T1, -T2, and -T3. The data show that 12 out of the 47 O-glycosylated sites were regulated, predominantly by GalNAc-T1. Using the glycoengineered cell lines for authentic EBOV propagation, we demonstrate the importance of O-linked glycan initiation and elongation for the production of viral particles and the titers of progeny virus. The mapped O-glycan positions and structures allowed to generate molecular dynamics simulations probing the largely unknown spatial arrangements of the mucin-like domain. The data highlight targeting GALNT1 or C1GALT1C1 as a possible way to modulate O-glycan density on EBOV GP for novel vaccine designs and tailored intervention approaches.IMPORTANCEEbola virus glycoprotein acquires its extensive glycan shield in the host cell, where it is decorated with N-linked glycans and mucin-type O-linked glycans. The latter is initiated by a family of polypeptide GalNAc-transferases that have different preferences for optimal peptide substrates resulting in a spectrum of both very selective and redundant substrates for each isoform. In this work, we map the exact locations of O-glycans on Ebola virus glycoprotein and identify subsets of sites preferentially initiated by one of the three key isoforms of GalNAc-Ts, demonstrating that each enzyme contributes to the glycan shield integrity. We further show that altering host O-glycosylation capacity has detrimental effects on Ebola virus replication, with both isoform-specific initiation and elongation playing a role. The combined structural and functional data highlight glycoengineered cell lines as useful tools for investigating molecular mechanisms imposed by specific glycans and for steering the immune responses in future vaccine designs.

Low expression of ELOVL6 may be involved in fat loss in white adipose tissue of cancer-associated cachexia

BackgroundCancer-associated cachexia (CAC) arises from malignant tumors and leads to a debilitating wasting syndrome. In the pathophysiology of CAC, the depletion of fat plays an important role. The mechanisms of CAC-induced fat loss include the enhancement of lipolysis, inhibition of lipogenesis, and browning of white adipose tissue (WAT). However, few lipid-metabolic enzymes have been reported to be involved in CAC. This study hypothesized that ELOVL6, a critical enzyme for the elongation of fatty acids, may be involved in fat loss in CAC.MethodsTranscriptome sequencing technology was used to identify CAC-related genes in the WAT of a CAC rodent model. Then, the expression level of ELOVL6 and the fatty acid composition were analyzed in a large clinical sample. Elovl6 was knocked down by siRNA in 3T3-L1 mouse preadipocytes to compare with wild-type 3T3-L1 cells treated with tumor cell conditioned medium.ResultsIn the WAT of patients with CAC, a significant decrease in the expression of ELOVL6 was found, which was linearly correlated with the extent of body mass reduction. Gas chromatographic analysis revealed an increase in palmitic acid (C16:0) and a decrease in linoleic acid (C18:2n-6) in these tissue samples. After treatment with tumor cell-conditioned medium, 3T3-L1 mouse preadipocytes showed a decrease in Elovl6 expression, and Elovl6-knockdown cells exhibited a reduction in preadipocyte differentiation and lipogenesis. Similarly, the knockdown of Elovl6 in 3T3-L1 cells resulted in a significant increase in palmitic acid (C16:0) and a marked decrease in oleic acid (C18:1n-9) content.ConclusionOverall, the expression of ELOVL6 was decreased in the WAT of CAC patients. Decreased expression of ELOVL6 might induce fat loss in CAC patients by potentially altering the fatty acid composition of adipocytes. These findings suggest that ELOVL6 may be used as a valuable biomarker for the early diagnosis of CAC and may hold promise as a target for future therapies.

Abstract PR007: Identifying targeted therapies for muscle invasive bladder cancer via STAG2 expression

Abstract High stromal antigen 2 (STAG2) protein expression is significantly associated with shorter overall and progression free survival of muscle invasive bladder cancer (MIBC) patients. Unfortunately, STAG2 itself is difficult to target as it functions as a transcriptional regulator and has no intrinsic enzymatic activity. Therefore, it becomes essential to identify downstream therapeutic vulnerabilities based on STAG2 expression. In this study, we accomplish this by employing a drug screen in isogenic STAG2 wild-type (WT) and knock-out (KO) MIBC cell lines, analyzing drug sensitivity of DepMap bladder cancer cell lines, and performing in vitro dose-response experiments based on our drug screen findings. To delineate changes in drug sensitivity due specifically to altered STAG2 expression, we used CRISPR-Cas9 to knock out STAG2 expression in T24 MIBC cells and generated two clonal STAG2 KO cell lines to compare to control. Then, we performed a drug screen with 312 drugs targeting oncogenic signaling pathways. We treated cells with drugs at 534 nM and read viability after 72 hours. To expand our drug screen findings to a larger group of cell lines and drugs, we further queried the DepMap database which includes drug sensitivity data for 4,868 drugs and approximately 2,000 cancer cell lines. We selected drug screened bladder cancer cell lines from DepMap and grouped them into ‘STAG2-high’ or ‘STAG2-low’ cohorts based on STAG2 mRNA expression. We then compared these two groups to identify if either group was more sensitive to drugs in the DepMap database. From these drug screen and DepMap analyses, we identified TAK-733 (MEKi), Berzosertib (ATRi), Prexasertib (CHKi), PI-103 (PI3Ki), and Rigosertib (PLKi) as candidate drugs that are differentially effective based on STAG2 expression level. We performed validation studies with these candidate drugs by treating matched STAG2 WT and KO T24 cells with each drug at concentrations ranging from 0.001 to 100 µM, then computationally modeled EC50 values in R. Cells with STAG2 KO were more sensitive to Berzosertib (T24 Control EC50: 1.78 µM, T24 KO EC50s: 1.03, 0.77 µM) compared to cells with WT STAG2 expression. Interestingly, cells with WT STAG2 expression were more sensitive to PI-103 (T24 Control EC50: 0.33 µM, T24 KO EC50s: 0.97, 1.41 µM) compared to cells with STAG2 KO. This suggests that STAG2 expressing cells may have a unique dependency on PI3K signaling, which has previously been reported in sarcoma cell lines. These findings lay the foundation for further investigation of Berzosertib as a targeted therapy for MIBC patients with mutated or low STAG2, and PI-103 for MIBC patients with high STAG2 who currently suffer worse clinical outcomes. Citation Format: Sarah R. Athans, Henry Withers, Zara Kazmierczak, Katerina Gurova, Anna Woloszynska. Identifying targeted therapies for muscle invasive bladder cancer via STAG2 expression [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR007.

NEK2 promotes TP53 ubiquitination to enhance the proliferation and migration of TP53 wild-type glioblastoma cells.

The most common primary malignant tumor in the adult brain is glioblastoma multiforme (GBM); however, its underlying pathogenic mechanism remains elusive. The never in mitosis (NIMA)-related kinase 2 (NEK2) has been closely associated with the prognosis of various malignancies. Nevertheless, the complete elucidation of NEK2's potential clinical value, particularly in glioma prognosis and development, remains lacking. U87MG and A172 glioblastoma cells were infected with sh-NEK2 lentivirus or oe-NEK2 plasmid to investigate the effect of NEK2 on cell proliferation, migration, and invasion. Cell viability was measured using CCK-8 and colony formation assays, while Transwell assay was utilized to assess cell migration and invasion. Protein expression levels were determined through western blot analysis. Additionally, CGGA and TCGA databases were used for bioinformatics analysis in order to examine the NEK2 expression. Through comprehensive bioinformatics analysis, we identified elevated mRNA expression levels of NEK2 in gliomas compared to normal tissues, which correlated with poor prognosis among glioma patients. Moreover, functional experiments revealed that silencing NEK2 suppressed glioma cell proliferation while overexpression of NEK2 promoted migration and invasion capabilities. Finally, our study uncovered that NEK2 regulates the malignant progression of TP53 wild-type glioblastoma by facilitating TP53 ubiquitination.

Decitabine cytotoxicity is promoted by dCMP deaminase DCTD and mitigated by SUMO-dependent E3 ligase TOPORS.

The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks(DNMT1-DPCs), leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with the identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.

Heterogeneous identity, stiffness and growth characterise the shoot apex of Arabidopsis stem cell mutants.

Stem cell homeostasis in the shoot apical meristem involves a core regulatory feedback loop between the signalling peptide CLAVATA3, produced in stem cells, and the transcription factor WUSCHEL, expressed in the underlying organising centre. clavata mutant meristems display massive overgrowth, which is thought to be caused by stem cell overproliferation, although it is unknown how uncontrolled stem cell divisions lead to this altered morphology. Here we reveal local buckling defects in mutant meristems, and use analytical models to show how mechanical properties and growth rates may contribute to the phenotype. Indeed, clavata meristems are mechanically more heterogeneous than the wild type, and also display regional growth heterogeneities. Furthermore, stereotypical wild-type meristem organisation is lost in mutants, in which cells simultaneously express distinct fate markers. Finally, cells in mutant meristems are auxin responsive, suggesting that they are functionally distinguishable from wild-type stem cells. Thus all benchmarks show that clavata meristem cells are different from wild-type stem cells, suggesting that overgrowth is caused by the disruption of a more complex regulatory framework that maintains distinct genetic and functional domains in the meristem.

Neratinib, a pan ERBB/HER inhibitor, restores sensitivity of PTEN-null, BRAFV600E melanoma to BRAF/MEK inhibition

IntroductionApproximately 50% of melanomas harbor an activating BRAFV600E mutation. Standard of care involves a combination of inhibitors targeting mutant BRAF and MEK1/2, the substrate for BRAF in the MAPK pathway. PTEN loss-of-function mutations occur in ~40% of BRAFV600E melanomas, resulting in increased PI3K/AKT activity that enhances resistance to BRAF/MEK combination inhibitor therapy.MethodsTo compare the response of PTEN null to PTEN wild-type cells in an isogenic background, CRISPR/Cas9 was used to knock out PTEN in a melanoma cell line that harbors a BRAFV600E mutation. RNA sequencing, functional kinome analysis, and drug synergy screening were employed in the context of BRAF/MEK inhibition.ResultsRNA sequencing and functional kinome analysis revealed that the loss of PTEN led to an induction of FOXD3 and an increase in expression of the FOXD3 target gene, ERBB3/HER3. Inhibition of BRAF and MEK1/2 in PTEN null, BRAFV600E cells dramatically induced the expression of ERBB3/HER3 relative to wild-type cells. A synergy screen of epigenetic modifiers and kinase inhibitors in combination with BRAFi/MEKi revealed that the pan ERBB/HER inhibitor, neratinib, could reverse the resistance observed in PTEN null, BRAFV600E cells.ConclusionsThe findings indicate that PTEN null BRAFV600E melanoma exhibits increased reliance on ERBB/HER signaling when treated with clinically approved BRAFi/MEKi combinations. Future studies are warranted to test neratinib reversal of BRAFi/MEKi resistance in patient melanomas expressing ERBB3/HER3 in combination with its dimerization partner ERBB2/HER2.

The clonal hematopoiesis mutation Jak2V617F aggravates endothelial injury and thrombosis in arteries with erosion-like intimas

BackgroundSuperficial plaque erosion causes many acute coronary syndromes. However, mechanisms of plaque erosion remain poorly understood, and we lack directed therapeutics for thrombotic complication. Human eroded plaques can harbor neutrophil extracellular traps (NETs) that propagate endothelial damage at experimental arterial lesions that recapitulate superficial erosion. Clonal Hematopoiesis of Indeterminate Potential (CHIP) denotes age-related clonal expansion of bone marrow-derived cells harboring somatic mutations in the absence of overt hematological disease. CHIP heightens the risk of cardiovascular disease, with the greatest increase seen in individuals with JAK2V617F. Neutrophils from mice and humans with JAK2V617F undergo NETosis more readily than Jak2WT (wild-type) cells. We hypothesized that JAK2V617F, by increasing propensity to NETosis, exacerbates aspects of superficial erosion. Methods and resultsWe generated Jak2V617F and Jak2WT mice with heterozygous Jak2V617F in myeloid cells. We induced areas of denuded endothelium that recapitulate features of superficial erosion and assessed endothelial integrity, cellular composition of the erosion, thrombosis rates, and response to ruxolitinib, a clinically available JAK1/2 inhibitor, in relation to genotype. Following experimental erosion, Jak2V617F mice have greater impairment of endothelial barrier function and increased rates of arterial thrombosis. Neointimas in Jak2V617F mice exhibit increased apoptosis, NETosis, and platelet recruitment. Jak2V617F mice treated with ruxolitinib show increased endothelial continuity and reduced apoptosis in the neointima comparable to levels in Jak2WT. ConclusionsThese observations provide new mechanistic insight into the pathophysiology of superficial erosion, the heightened risk for myocardial infarction in JAK2V617F CHIP, and point the way to personalized therapeutics based on CHIP status.

Long-term hematopoietic transfer of the anti-cancer and lifespan-extending capabilities of a genetically engineered blood system by transplantation of bone marrow mononuclear cells

A causal relationship exists among the aging process, organ decay and disfunction, and the occurrence of various diseases including cancer. A genetically engineered mouse model, termed Klf1K74R/K74R or Klf1(K74R), carrying mutation on the well-conserved sumoylation site of the hematopoietic transcription factor KLF1/EKLF has been generated that possesses extended lifespan and healthy characteristics, including cancer resistance. We show that the healthy longevity characteristics of the Klf1(K74R) mice, as exemplified by their higher anti-cancer capability, are likely gender-, age-, and genetic background-independent. Significantly, the anti-cancer capability, in particular that against melanoma as well as hepatocellular carcinoma, and lifespan-extending property of Klf1(K74R) mice, could be transferred to wild-type mice via transplantation of their bone marrow mononuclear cells at a young age of the latter. Furthermore, NK(K74R) cells carry higher in vitro cancer cell-killing ability than wild-type NK cells. Targeted/global gene expression profiling analysis has identified changes in the expression of specific proteins, including the immune checkpoint factors PDCD and CD274, and cellular pathways in the leukocytes of the Klf1(K74R) that are in the directions of anti-cancer and/or anti-aging. This study demonstrates the feasibility of developing a transferable hematopoietic/blood system for long-term anti-cancer and, potentially, for anti-aging.

Long-term hematopoietic transfer of the anti-cancer and lifespan-extending capabilities of a genetically engineered blood system by transplantation of bone marrow mononuclear cells.

A causal relationship exists among the aging process, organ decay and disfunction, and the occurrence of various diseases including cancer. A genetically engineered mouse model, termed Klf1K74R/K74R or Klf1(K74R), carrying mutation on the well-conserved sumoylation site of the hematopoietic transcription factor KLF1/EKLF has been generated that possesses extended lifespan and healthy characteristics, including cancer resistance. We show that the healthy longevity characteristics of the Klf1(K74R) mice, as exemplified by their higher anti-cancer capability, are likely gender-, age-, and genetic background-independent. Significantly, the anti-cancer capability, in particular that against melanoma as well as hepatocellular carcinoma, and lifespan-extending property of Klf1(K74R) mice, could be transferred to wild-type mice via transplantation of their bone marrow mononuclear cells at a young age of the latter. Furthermore, NK(K74R) cells carry higher in vitro cancer cell-killing ability than wild-type NK cells. Targeted/global gene expression profiling analysis has identified changes in the expression of specific proteins, including the immune checkpoint factors PDCD and CD274, and cellular pathways in the leukocytes of the Klf1(K74R) that are in the directions of anti-cancer and/or anti-aging. This study demonstrates the feasibility of developing a transferable hematopoietic/blood system for long-term anti-cancer and, potentially, for anti-aging.

Fisetin and/or capecitabine causes changes in apoptosis pathways in capecitabine-resistant colorectal cancer cell lines.

Capecitabine is recommended as one of the first-line chemotherapy treatments for advanced or metastatic colorectal cancer. Researches have been conducted on capecitabine's impact on the viability of human colon cancer cells and its potential to induce apoptosis. However, even in cases initially responsive to treatment, the development of acquired resistance significantly limits its efficacy. Challenges still exist in effectively treating patients with chemotherapy, and developing new cytotoxic drugs is hindered by drug resistance. Fisetin alters the cell cycle, inducing apoptosis, inhibiting cancer cell proliferation, and enhancing the therapeutic effectiveness of chemotherapy drugs. This work aims to create a plan for reversing capecitabine resistance. For this purpose, the role of capecitabine and/or fisetin combinations in cell proliferation and apoptosis has been determined in both wild-type and capecitabine-resistant HT29 cells (CR/HT29). We developed capecitabine-resistant cell line from wild-type HT29 cells. This study demonstrated the effects of capecitabine, fisetin, and their combinations on both resistant and wild-type cells through experiments including cell survival skills, cell proliferation, wound healing, colony formation, hoechst staining, and western blot analysis.We established capecitabine-resistant cell lines. P-gp expression increased in CR/HT29 cells. Capecitabine effects on a CR/HT29 cells less than wild-type HT29 cells. The combination of fisetin and capecitabine in cell proliferation caused greater reductions in wild-type HT29 cells than in capecitabine-resistant cells. Fisetin has also additive effects on the apoptotic pathway in CR/HT29 cells.This study provides new perspectives on the combination of capecitabine and/or flavonoid treatment in resistant cells.

Semiconservative transmission of DNA N6-adenine methylation in a unicellular eukaryote.

While DNA N6-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has generated great interest recently. Biochemical and genetic evidence supports that AMT1, a MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, 6mA transmission mechanism remains to be elucidated. Taking advantage of Single Molecule Real-Time Circular Consensus Sequencing (SMRT CCS), here we provide definitive evidence for semiconservative transmission of 6mA in Tetrahymena thermophila In wild-type (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); after DNA replication, hemi-methylation (hemi-6mApT) is transiently present on the parental strand, opposite to the daughter strand readily labeled by 5-bromo-2'-deoxyuridine (BrdU). In ΔAMT1 cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and processive, while de novo methylation in ΔAMT1 cells is slow and sporadic. In Tetrahymena, regularly spaced 6mA clusters coincide with linker DNA of nucleosomes arrayed in the gene body. Importantly, in vitro methylation of human chromatin by reconstituted AMT1 complex recapitulates preferential targeting of hemi-6mApT sites in linker DNA, supporting AMT1's intrinsic and autonomous role in maintenance methylation. We conclude that 6mA is transmitted by a semiconservative mechanism: full-6mApT is split by DNA replication into hemi-6mApT, which is restored to full-6mApT by AMT1-dependent maintenance methylation. Our study dissects AMT1-dependent maintenance methylation and AMT1-independent de novo methylation, reveals a 6mA transmission pathway with striking similarity to 5-methyl cytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.