Cytoskeleton Organization Research Articles

Triploidization of rainbow trout affects proteins related to ovary development and reproductive activity

Rainbow trout are characterized by high amenability to chromosome manipulations during early development to produce androgens, gynogens, or sterile triploids, which are relevant in aquaculture. An understanding of the mechanisms affecting oogenesis and those resulting in diminished gonadal development in triploids is an important area of scientific study. Therefore, the aim of this study was to compare the ovary proteome from triploid (3 N) and diploid (2 N) rainbow trout females using tandem mass tag (TMT) peptide labelling coupled with liquid chromatography–mass spectrometry (LC–MS/MS) to identify differentially expressed proteins (DEPs) between 2 N and 3 N. The potential biological role of DEPs was also explored using comparative functional gene set enrichment to designate the potential protein markers of 3 N sterility. We identified a total of 3899 DEPs (q < 0.05), including 956 downregulated and 2943 upregulated proteins in 3 N ovaries compared to 2 N ovaries. The identified clusters for downregulated proteins in 3 N included lipid metabolic process, hormone biosynthesis, transport and exocytosis, cell junction organization and sperm-egg recognition, which can result in (i) the disruption of lipid, steroid hormone and vitellogenesis processes, (ii) impaired amino acid uptake, (iii) defects in cell–cell junctions reflecting tissue abnormalities and aberrant gonadal morphology, and (iv) impaired functions related to maintaining the proper structure of the vitelline envelope, oocyte formation and fertilization competence. With respect to upregulated proteins in 3 N ovaries, RNA processing, cell cycle, cytoskeleton organization and regulation of metabolic process were the primary clusters, which may reflect (i) the disturbance to RNA processing responsible for impairment of ovary development in triploid rainbow trout, (ii) disruption of spindle formation, incorrect chromosome alignment and segregation during meiotic division, (iii) increase in regulatory mechanism of apoptosis, which in consequence can provoke abnormal gonadal development and sterility, and (iv) enhanced ubiquitin–proteasome system governing aberrant gonadal morphology in 3 N females and oocyte maturation. The obtained results provide an accurate portrait of molecular changes in rainbow trout ovaries in response to triploidization and may contribute to a better understanding of the mechanisms responsible for impaired oogenesis in rainbow trout triploid females, which in turn leads to their sterility. Data are available via ProteomeXchange with identifier PXD034492.

Neoadjuvant chemotherapy is associated with an altered metabolic profile and increased cancer stemness in patients with pancreatic ductal adenocarcinoma.

The modest clinical benefits of neoadjuvant chemotherapy (NAT) in pancreatic ductal adenocarcinoma (PDAC) are associated with a lack of robust data on treatment-induced changes in the tumor. To this end, comparative proteomic profiling of tumor tissue samples from treatment-naïve (TN, n=20) and NAT-treated (n=22) PDACs was performed. Differentially expressed proteins were identified and correlation with overall survival (OS) was performed. Tumors were also examined for histopathological changes and expression of cancer stem cell (CSC) markers. Serum from 33 matched patients was analyzed for metabolic markers. Cytotoxicity, proliferation, and expression of CSC markers were assessed in chemoresistant Panc-1 and Mia PaCa-2 cells. Of the 2265 proteins identified, 227 and 144 proteins showed significantly altered expression and differential phosphorylation, respectively, in NAT compared with TN samples. The majority of these were metabolism-related proteins, and 14 of these correlated moderately with OS. NAT-treated tumors and chemoresistant cancer cells showed increased expression of CSC markers. Serum ALDH1A1 was higher in NAT compared with TN. Differentially phosphorylated proteins were mainly involved in cytoskeleton organization, cell locomotion, motility, and migration, and 17 of these showed a strong positive correlation with OS. This study provides evidence of the effects of NAT on PDAC metabolism at both the tumor and the systemic levels. NAT-treated tumors showed significantly lower expression of metabolic proteins, and patients who underwent NAT showed reduced serum lactate and high-density lipoprotein-cholesterol. Lastly, cancer cells that survived cytotoxic treatment expressed higher CSC markers, both in vivo and in vitro.

New Insights into the Mechanism of Antibacterial Action of Synthetic Peptide Mo-CBP3-PepI against Klebsiella pneumoniae.

Klebsiella pneumoniae is a multidrug-resistant opportunistic human pathogen related to various infections. As such, synthetic peptides have emerged as potential alternative molecules. Mo-CBP3-PepI has presented great activity against K. pneumoniae by presenting an MIC50 at a very low concentration (31.25 µg mL-1). Here, fluorescence microscopy and proteomic analysis revealed the alteration in cell membrane permeability, ROS overproduction, and protein profile of K. pneumoniae cells treated with Mo-CBP3-PepI. Mo-CBP3-PepI led to ROS overaccumulation and membrane pore formation in K. pneumoniae cells. Furthermore, the proteomic analysis highlighted changes in essential metabolic pathways. For example, after treatment of K. pneumoniae cells with Mo-CBP3-PepI, a reduction in the abundance of protein related to DNA and protein metabolism, cytoskeleton and cell wall organization, redox metabolism, regulation factors, ribosomal proteins, and resistance to antibiotics was seen. The reduction in proteins involved in vital processes for cell life, such as DNA repair, cell wall turnover, and protein turnover, results in the accumulation of ROS, driving the cell to death. Our findings indicated that Mo-CBP3-PepI might have mechanisms of action against K. pneumoniae cells, mitigating the development of resistance and thus being a potent molecule to be employed in producing new drugs against K. pneumoniae infections.

Organization and dynamics of cross-linked actin filaments in confined environments

The organization of the actin cytoskeleton is impacted by the interplay between physical confinement, features of cross-linking proteins, and deformations of semiflexible actin filaments. Some cross-linking proteins preferentially bind filaments in parallel, although others bind more indiscriminately. However, a quantitative understanding of how the mode of binding influences the assembly of actin networks in confined environments is lacking. Here we employ coarse-grained computer simulations to study the dynamics and organization of semiflexible actin filaments in confined regions upon the addition of cross-linkers. We characterize how the emergent behavior is influenced by the system shape, the number and type of cross-linking proteins, and the length of filaments. Structures include isolated clusters of filaments, highly connected filament bundles, and networks of interconnected bundles and loops. Elongation of one dimension of the system promotes the formation of long bundles that align with the elongated axis. Dynamics are governed by rapid cross-linking into aggregates, followed by a slower change in their shape and connectivity. Cross-linking decreases the average bending energy of short or sparsely connected filaments by suppressing shape fluctuations. However, it increases the average bending energy in highly connected networks because filament bundles become deformed, and small numbers of filaments exhibit long-lived, highly unfavorable configurations. Indiscriminate cross-linking promotes the formation of high-energy configurations due to the increased likelihood of unfavorable, difficult-to-relax configurations at early times. Taken together, this work demonstrates physical mechanisms by which cross-linker binding and physical confinement impact the emergent behavior of actin networks, which is relevant both in cells and in synthetic environments.

Placental macrophage responses to viral and bacterial ligands and the influence of fetal sex.

Bacterial and viral infections of the placenta are associated with inflammation and adverse pregnancy outcomes. Hofbauer cells (HBCs) are fetal-origin macrophages in the placenta, proposed to protect the fetus from vertical pathogen transmission. We performed quantitative proteomics on term HBCs under resting conditions and following exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs), and investigated the contribution of fetal sex. Resting HBCs expressed proteins pertinent to macrophage function, including chemokines, cytokines, Toll-like receptors, and major histocompatibility complex class I and II molecules. HBCs mounted divergent responses to bacterial versus viral PAMPs but exhibited protein expression changes suggestive of a more pro-inflammatoryphenotype. A comparison between male and female HBCs showed that the latter mounted a stronger and wider response. Here, we provide a comprehensive understanding of the sex-dependent responses of placental macrophages to infectious triggers, which were primarily associated with lipid metabolism in males and cytoskeleton organization in females.

ROCK inhibitors in glaucoma medical treatment

AbstractRho kinase inhibitors are a novel advancement in glaucoma medical treatment and have been recently approved for instruction in the market in fixed combination with the prostaglandin analogue Latanoprost. They have a novel mechanism of action based on their ability to improve humour aqueous outflow through the conventional trabecular meshwork pathway.Rho family proteins are GTPases that localize in the intracellular side of plasma membrane and are activated by several plasma membrane signal receptors, including TGF‐β and hormones.Activated Rho then activates Rho‐associated protein kinases which further transmit the stimulus to downstream effectors. The whole pathway is involved in several cellular activities, including actin cytoskeleton organization, cell adhesion and motility, proliferation and apoptosis, and remodelling of the ECM. ROCKs mediate pro‐fibrotic processes in many tissues and pathological conditions. In cultured human TM cells, exposure to a ROCK inhibitor caused retraction and rounding of cell bodies, as well as disruption of actin bundles and impaired focal adhesion formation. ROCKs have been associated when administered topically to a reduction in episcleral venous pressure that might contribute to their ocular hypotensive effects.MERCURY‐3, was a prospective, randomized, double‐masked, active‐controlled study to assess the efficacy, safety, and tolerability of the ROCK inhibitor netarsudil 0.02% in fixed combination with latanoprost 0.005% compared with the fixed combination of bimatoprost 0.03%/timolol 0.5%.The results of the study showed non inferiority of the netarsudil fixed combination despite a slightly worse local tolerability profile.Current management approaches aim to reduce IOP but do not tackle the principal site of increased outflow resistance that is the TM. ROCK inhibition shows for the first time the ability for directly modulating the TM permeability and represents a new clinical treatment for glaucoma.

Transcriptome profile and its partly verification of human hepatocellular carcinoma cells exposed to Yuzhizi () seed extract.

To investigate the efficacy of Yuzhizi seed extract (FAQSE) on inhibiting the proliferation of hepatocellular carcinoma (HCC) cells in vitro and to explore the anti-HCC action mechanism of FAQSE. Human HCC HepG2 and Huh7 cells were used to investigate the anti-HCC effect of FAQSE. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) method was used to measure cell viability. Affymetrix microarray was adopted to detect the expression of transcriptome. The differentially expressed genes (DEGs) of each cell line were identified. For co-DEGs of both cell lines, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were enriched using the Database for Annotation, Visualization and Integrated Discovery (DAVID), and the network analysis of protein-protein interaction (PPI) was mapped using the Retrieval of Interacting Genes/Proteins (STRING) and Cytoscape software. Some important genes in the PPI network of co-DEGs were selected to verify by quantitative real-time reverse transcription-polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay. FAQSE decreased the viability of HepG2 and Huh7 cells. There were 211 co-upregulated and 86 co-downregualted genes in both cell lines after FAQSE treatment. The enriched GO terms of co-upregulated DEGs were primarily involved cell-cell adhesion, viral process, transcription initiation from RNA polymerase II promoter, positive regulation of transcription from RNA polymerase II promoter and actin cytoskeleton organization. The GO terms of co-downregulated DEGs were mainly enriched in the processes of SRP-dependent cotranslational protein targeting to membrane, viral transcription, nuclear-transcribed mRNA catabolic process, nonsense-mediated decay, translational initiation and rRNA processing. Main KEGG pathways of co-upregulated DEGs were endocytosis, glutathione metabolism, protein processing in endoplasmic reticulum, synaptic vesicle cycle and lysosome. The major KEGG pathways of co-downregulated DEGs were ribosome, biosynthesis of amino acids, arginine and proline metabolism, systemic lupus erythematosus and complement and coagulation cascades. The top 10 co-DEGs with high hub nodes in STRING analysis were ribosomal protein S27a, transferrin, ribosomal protein S20, ribosomal protein L9, protein phosphatase 2 regulatory subunit B alpha, transthyretin, thioredoxin reductase 1, ribosomal protein L3, ribophorin I and ribosomal protein L24. Alpha-fetoprotein (AFP) was also co-downregulated and contained in the PPI network. The mRNA and protein expression of most verified genes was consistent with the results of co-DEGs analysis. And the AFP level was significantly reduced after FAQSE treatment. A series of genes and pathways of HepG2 and Huh7 cells were changed after FAQSE treatment, which might be the targets of FAQSE against HCC and worthy of further study. AFP might be important one of them.

Investigating the Function of Human Jumping Translocation Breakpoint Protein (hJTB) and Its Interacting Partners through In-Solution Proteomics of MCF7 Cells.

Human jumping translocation breakpoint (hJTB) gene is located on chromosome 1q21 and is involved in unbalanced translocation in many types of cancer. JTB protein is ubiquitously present in normal cells but it is found to be overexpressed or downregulated in various types of cancer cells, where this protein and its isoforms promote mitochondrial dysfunction, resistance to apoptosis, genomic instability, proliferation, invasion and metastasis. Hence, JTB could be a tumor biomarker for different types of cancer, such as breast cancer (BC), and could be used as a drug target for therapy. However, the functions of the protein or the pathways through which it increases cell proliferation and invasiveness of cancer cells are not well-known. Therefore, we aim to investigate the functions of JTB by using in-solution digestion-based cellular proteomics of control and upregulated and downregulated JTB protein in MCF7 breast cancer cell line, taking account that in-solution digestion-based proteomics experiments are complementary to the initial in-gel based ones. Proteomics analysis allows investigation of protein dysregulation patterns that indicate the function of the protein and its interacting partners, as well as the pathways and biological processes through which it functions. We concluded that JTB dysregulation increases the epithelial-mesenchymal transition (EMT) potential and cell proliferation, harnessing cytoskeleton organization, apical junctional complex, metabolic reprogramming, and cellular proteostasis. Deregulated JTB expression was found to be associated with several proteins involved in mitochondrial organization and function, oxidative stress (OS), apoptosis, and interferon alpha and gamma signaling. Consistent and complementary to our previous results emerged by using in-gel based proteomics of transfected MCF7 cells, JTB-related proteins that are overexpressed in this experiment suggest the development of a more aggressive phenotype and behavior for this luminal type A non-invasive/poor-invasive human BC cell line that does not usually migrate or invade compared with the highly metastatic MDA-MB-231 cells. This more aggressive phenotype of MCF7 cells related to JTB dysregulation and detected by both in-gel and in-solution proteomics could be promoted by synergistic upregulation of EMT, Mitotic spindle and Fatty acid metabolism pathways. However, in both JTB dysregulated conditions, several downregulated JTB-interacting proteins predominantly sustain antitumor activities, attenuating some of the aggressive phenotypical and behavioral traits promoted by the overexpressed JTB-related partners.

ZO-1 Guides Tight Junction Assembly and Epithelial Morphogenesis via Cytoskeletal Tension-Dependent and -Independent Functions

Formation and maintenance of tissue barriers require the coordination of cell mechanics and cell-cell junction assembly. Here, we combined methods to modulate ECM stiffness and to measure mechanical forces on adhesion complexes to investigate how tight junctions regulate cell mechanics and epithelial morphogenesis. We found that depletion of the tight junction adaptor ZO-1 disrupted junction assembly and morphogenesis in an ECM stiffness-dependent manner and led to a stiffness-dependant reorganisation of active myosin. Both junction formation and morphogenesis were rescued by inhibition of actomyosin contractility. ZO-1 depletion also impacted mechanical tension at cell-matrix and E-cadherin-based cell-cell adhesions. The effect on E-cadherin also depended on ECM stiffness and correlated with effects of ECM stiffness on actin cytoskeleton organisation. However, ZO-1 knockout also revealed tension-independent functions of ZO-1. ZO-1-deficient cells could assemble functional barriers at low tension, but their tight junctions remained corrupted with strongly reduced and discontinuous recruitment of junctional components. Our results thus reveal that reciprocal regulation between ZO-1 and cell mechanics controls tight junction assembly and epithelial morphogenesis, and that, in a second, tension-independent step, ZO-1 is required to assemble morphologically and structurally fully assembled and functionally normal tight junctions.

Efficacy of low-dose hCG on FET cycle in patients with recurrent implantation failure.

To study patients' new treatment methods and mechanisms of repeated implantation failure. A retrospective study. In vitro fertilization (IVF) unit in a Three-A hospital. Ninety-three patients with repeated implantation failure in IVF and embryo transfer. the luteal phase support. According to whether human chorionic gonadotropin(HCG) was added, the two groups were divided into an observation group and a control group, and the clinical outcomes of the two groups were compared. Furthermore, 20 patients were selected for whole exome sequencing to investigate the mechanism. The observation group's clinical pregnancy rate and live birth rate were significantly higher than those in the control group (P=0.004). Functional enrichment analysis showed that these genes were significantly enriched in embryo implantation or endometrial receptivity processes, such as microtubule-based movement, NABA CORE MATRISOME, superoxide anion generation, protein localization to vacuole, extracellular matrix organization, fertilization, microtubule-based transport, cell junction organization, microtubule cytoskeleton organization. Furthermore, variants detected in these pathway genes were missense mutations that affect the protein's biological activity but do not effectuate its inactivation. Adding HCG in the luteal phase might improve the clinical pregnancy and live birth rates in RIF patients. The potential pathogenesis of RIF genetic level may be caused by microtubule-based movement, extracellular matrix organization, and the Superoxide Anion generation pathway.

Rheological comparison between control and Dupuytren fibroblasts when plated in circular micropatterns using atomic force microscopy

In tissue, cells are obliged to confine and adapt to a specific geometric shape due to the surrounding environmental constraints. Under healthy conditions, fibroblasts present an elongated shape; however, changes in biochemical and physical properties of the extracellular matrix could distort the cell shape, inducing a pathological state. We have studied fibroblasts’ mechanical behavior under circular geometrical constraints. Circular micropatterns force fibroblasts to acquire a different shape from that of a healthy tissue, inducing a possible pathological condition. In total, three different fibroblast types from Dupuytren’s disorder, all obtained from the same patient, were confined in circular-shaped micropatterns of three different diameters (25, 35, and 45 µm), and mechanical properties were evaluated using an atomic force microscope (AFM). We found that control fibroblast mechanics (apparent Young’s modulus) increases with the increasing pattern diameter and comes together with a decrease in cell height and in loss tangent, translated into a more solid-like behavior. We hypothesize that these results resemble the transition toward the myofibroblast phenotype, ameliorating cytoskeleton formation and organization and enhancing cell contraction. Scar and Dupuytren fibroblasts did not display major changes in cell mechanics and cell height when changing the pattern diameter, suggesting that they are less affected by physical changes in the environment as they can adapt their shape to the geometrical dimensions. Therefore, our findings demonstrate that combining micropatterning and AFM measurements provides a powerful tool to study cell mechanics inducing constraints onto the cell, thus mimicking certain aspects of the tissue environment in both healthy and pathological states.

3D Microfluidic System for Evaluating Inhibitory Effect of Chinese Herbal Medicine Oldenlandia diffusa on Human Malignant Glioma Invasion Combined with Network Pharmacology Analysis.

To investigate the anti-invasion efficacy of the ethanol extract of Oldenlandia diffusa Will. (EEOD) on a three-dimensional (3D) human malignant glioma (MG) cell invasion and perfusion model based on microfluidic chip culture and the possible mechanism of action of Oldenlandia diffusa Will. (OD). The comprehensive pharmacodynamic analysis method in this study was based on microfluidic chip 3D cell perfusion culture technology, and the action mechanism of Chinese medicine (CM) on human MG cells was investigated through network pharmacology analysis. First, the components of EEOD were analyzed by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Then, cell viability and apoptosis were assessed to determine the optimum concentration of EEOD for invasion experiments, and two-dimensional (2D) migration and invasion abilities of U87 and U251 MG cells were evaluated using scratch wound and Transwell assays. The possible mechanism underlying the effects of EEOD on glioma was analyzed through a network pharmacology approach. Thirty-five compounds of EEOD were detected by UPLC-Q-TOF/MS. EEOD suppressed the viability of MG cells, promoted their apoptosis, and inhibited their migratory and invasive potentials (all P<0.05). Network pharmacology analysis showed that OD inhibited the invasion of MG cells by directly regulating MAPK and Wnt pathways through MAPK, EGFR, MYC, GSK3B, and other targets. The anti-invasion effect of OD was also found to be related to the indirect regulation of microtubule cytoskeleton organization. ]EEOD could inhibit the invasion of human MG cells, and the anti-invasion mechanism of OD might be regulating MAPK and Wnt signaling pathways and microtubule cytoskeleton organization.

MiR-708-5p Regulates Myoblast Proliferation and Differentiation.

MicroRNAs (miRNAs) are key regulators involved in the myogenic process in skeletal muscles. miR-708-5p plays an important role in various biochemical and physiological processes, but its function in skeletal myogenesis remain unclear. In this study, we first explored the effects of miR-708-5p on C2C12 proliferation and differentiation by overexpression and interference experiments. Then, we predicted the target genes of miR-708-5p and analyzed their function. We found that miR-708-5p was gradually increased during myoblast differentiation. Overexpression of miR-708-5p significantly inhibited cell proliferation and promoted the differentiation of myoblasts. A total of 253 target genes were predicted using a bioinformatics approach. These genes were significantly enriched in muscle growth-related GO terms and KEGG pathways, such as actin filament organization, actin cytoskeleton organization, PI3K-Akt pathway, insulin pathway, and Jak-STAT pathway. Among them, Pik3ca, Pik3r3, and Irs1 were considered to be the key target genes of miR-708-5p. To sum up, miR-708-5p inhibited C2C12 cells proliferation and promoted C2C12 cells differentiation. Its target genes significantly enriched in GO terms and KEGG pathways related to the development and growth of muscle. Our results provided a basis for studies on the function and mechanism of miR-708-5p regulating skeletal muscle growth and development.

Requirement for PINCH in skeletal myoblast differentiation

PINCH, an adaptor of focal adhesion complex, plays essential roles in multiple cellular processes and organogenesis. Here, we ablated PINCH1 or both of PINCH1 and PINCH2 in skeletal muscle progenitors using MyoD-Cre. Double ablation of PINCH1 and PINCH2 resulted in early postnatal lethality with reduced size of skeletal muscles and detachment of diaphragm muscles from the body wall. PINCH mutant myofibers failed to undergo multinucleation and exhibited disrupted sarcomere structures. The mutant myoblasts in culture were able to adhere to newly formed myotubes but impeded in cell fusion and subsequent sarcomere genesis and cytoskeleton organization. Consistent with this, expression of integrin β1 and some cytoskeleton proteins and phosphorylation of ERK and AKT were significantly reduced in PINCH mutants. However, N-cadherin was correctly expressed at cell adhesion sites in PINCH mutant cells, suggesting that PINCH may play a direct role in myoblast fusion. Expression of MRF4, the most highly expressed myogenic factor at late stages of myogenesis, was abolished in PINCH mutants that could contribute to observed phenotypes. In addition, mice with PINCH1 being ablated in myogenic progenitors exhibited only mild centronuclear myopathic changes, suggesting a compensatory role of PINCH2 in myogenic differentiation. Our results revealed a critical role of PINCH proteins in myogenic differentiation.

Regulation of actin cytoskeleton via photolithographic micropatterning

Actin cytoskeleton plays crucial roles in various cellular functions. Extracellular matrix (ECM) can modulate cell morphology by remodeling the internal cytoskeleton. To define how geometry of ECM regulates the organization of actin cytoskeleton, we plated individual NIH 3T3 cells on micropatterned substrates with distinct shapes and sizes. It was found that the stress fibers could form along the nonadhesive edges of T-shaped pattern, but were absent from the opening edge of V-shaped pattern, indicating that the organization of actin cytoskeleton was dependent on the mechanical environment. Furthermore, a secondary actin ring was observed on 50[Formula: see text][Formula: see text]m circular pattern while did not appear on 30[Formula: see text][Formula: see text]m and 40[Formula: see text][Formula: see text]m pattern, showing a size-dependent organization of actin cytoskeleton. Finally, osteoblasts, MDCK and A549 cells exhibited distinct organization of actin cytoskeleton on T-shaped pattern, suggesting a cell-type specificity in arrangement of actin cytoskeleton. Together, our findings brought novel insight into the organization of actin cytoskeleton on micropatterned environments.

Aging-Related Gene Expression Signatures Define Likelihood of Disease Progression in Patients with Lower-Risk Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a heterogeneous clonal bone marrow disorders occurring mostly in the elderly. The Revised International Prognostic System (IPSS-R) groups MDS patients according to the risk of transformation into acute myeloid leukemia. For treatment decision-making, patients are usually divided into two groups: lower- and higher-risk with the cutpoint of 3.5 according to the IPSS-R. However, part of lower-risk MDS patients (LR-MDS) progresses rapidly. Recently, we demonstrated that DNA damage response (DDR) activation and the ensuing senescence form an anti-tumor barrier in CD34+ cells of LR-MDS patients, and that this barrier can be breached by RUNX1 mutations (Kaisrlikova M, et al., Leukemia. 2022; 36:1898). As an extension, we analyzed transcriptomes of LR-MDS patients regardless of their driver mutation, with and without rapid progression (within 2 years of diagnosis), to determine molecular mechanisms underlying the progression. Because MDS is associated with aging, and many anti-tumor barrier mechanisms (DDR, checkpoint activation, senescence) lead to a pro-aging phenotype, we questioned whether aging-related gene expression signatures define the likelihood of disease progression in patients with LR-MDS. We performed RNA-seq on CD34+ cells of 61 LR-MDS diagnostic samples (53 patients without rapid progression (NRP) and 8 patients with rapid progression (RP)) with a median age of 65 years. The differential expression analysis showed 1,701 up- and 1,569 downregulated genes in NRP (FDR<0.01). According to the GO Biological Processes (GO BP) and REACTOME databases, the most significantly upregulated pathways were related to macromolecular metabolic processes such as DNA repair, replication, protein metabolism, cell cycle, chromosome organization, including regulation of gene expression, and telomere organization (Fig. 1). The pathways of rRNA expression regulation, splicing, and RNA metabolism were deregulated as well as the pathways related to apoptosis, cellular senescence and cellular response to stress, immune response, and mitochondrial metabolism. Furthermore, we found deregulation of crucial signaling pathways mediated by NOTCH, RHO GTPases, NF-kB, MAPK, b-catenin, and WNT. Although 1,569 genes were upregulated in RP, only seventeen GO BP terms consisted of regulation of metabolic processes, cytoskeleton organization, phosphorylation, and signal transduction were upregulated. The NRP results are consistent with the hallmarks of aging (López-Otín C, et al., Cell. 2013; 153:1194). In GSEA, 36/37 and 120/143 gene sets were upregulated in NRP for the datasets consisting of gene sets chosen according to the keyword 'aging' and 'stress', respectively (Fig. 2). Among them, gene sets associated with replication stress, DNA damage, oxidative stress and unfolded protein response were significant (FDR<0.1, ІNESІ>1.7). Abnormal splicing is associated with cellular aging. Therefore, we conducted a differential alternative splicing analysis using rMATS. We found significant differences in 1,356 alternative splicing events (FDR<0.05, absolute inclusion level difference>10%) between NRP and RP. Finally, we performed analysis of healthy controls (n=7) and age-matched NRP (NRP-age) (n=13) samples. We observed 70 up- and 209 downregulated genes in NRP-age (FDR<0.05). Only the splicing pathways were upregulated and the immune pathways downregulated. In GSEA, no gene sets were significant (FDR<0.1) for aging and stress datasets. To conclude, these results show increased signatures of aging in CD34+ cells from NRP, which correspond to an increased level of the intrinsic ability of CD34+ cells to suppress malignant progression through anti-tumor barrier activation. This is consistent with the current understanding that most aging phenotypes are known to be the products of DDR and tumor suppression. Based on our data, we hypothesize that the progression of LR-MDS patients represents a cell-autonomously and perhaps also non-cell-autonomously-mediated failure of appropriate cellular stress response mechanisms. Our data also show similarity at the transcriptome level between age-matched NRP and healthy controls. Supported by AZV (NV18-03-00227) and (NU21-03-00565), GA CR (N20-19162S), and MH CZ-DRO (UHKT, 00023736). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Biomarkers of Clinical Resistance to Decitabine and Venetoclax Therapy Inferred By Transcriptome and Methylation Analyses

The combination of the selective BCL-2 inhibitor venetoclax (VEN) with the hypomethylating agent (HMA) decitabine (DEC) is highly effective in treating older ineligible for intensive chemotherapy AML patients. However, approximately 15-34% of newly diagnosed (ND) and 60% of relapsed/refractory (R/R) patients fail to achieve complete remission (CR) (DiNardo et al. New Engl J Med. 2020, DiNardo et al. Lancet Haematol. 2020). Factors that reduce efficacy remain to be elucidated. To dissect underlying mechanisms of HMA-VEN resistance, we performed RNA-seq utilizing primary samples from patients treated on a prospective clinical trial of 10-day DEC with VEN (NCT03404193). Previously, we reported that alterations of transcriptome signatures of energy-metabolism and cell migration/adhesion pathways were upregulated in primary resistant ND group, and immune-system-associated genes were downregulated in R/R group (Yamatani et al. ASH 2021). This study investigated transcriptomic and DNA methylation signatures associated with DEC10-VEN response or resistance in ND and R/R AML. Because DEC was reported to strongly induce interferon-stimulated genes (ISGs) through gene methylation modifications, we focused on 116 ISGs. An integrated analysis of DNA methylation status around the transcription start site (TSS) was performed using the Infinium methylated EPIC array (Illumina). Forty-four pre-treatment samples from 32 patients with AML from DEC-VEN clinical trial, ND patient group (CR/CRi 14; non-responder, NR 11) and R/R patient group (CR/CRi 6; NR 13), were analyzed. First, we performed the comparative analysis of NR vs CRs. In the ND group, NR cases showed significantly higher gene expression and lower TSS methylation of an urea cycle enzyme, ASS1 (Argininosuccinate Synthase 1) with RNA-seq Log2 fold-change (FC) 3.6 and methylation array Δβ (differential methylation β-value) -0.11, p < 0.05, and of an actin-based molecular motor, MYO1C (Myosin 1C), Log2FC 2.5, Δβ = -0.18, p< 0.05, than in CR cases. In the R/R group, gene expression levels of seven ISGs, including CCL3 (C-C motif chemokine 3), were significantly lower in NR than in CRs, with no difference in methylation status. Next, changes in gene expression levels and TSS methylation status after DEC-VEN treatment were investigated in non-responders in ND group (pre 19, post 12) and R/R group (pre 16, post 4). After DEC-VEN treatment, PPARG (Peroxisome Proliferator-Activated Receptor γ), a transcription factor that activates fatty acid metabolism and negatively regulates subsets of IFN-γ target genes, was significantly upregulated along with its TSS demethylation both in ND (Log2FC 4.9, Δβ = -0.14, p < 0.05), and R/R groups (Log2FC 3.7, Δβ = -0.06, p < 0.05). CDC42EP4, an effector protein of CDC42 involved in the organization of the actin cytoskeleton, was upregulated after the treatment in the R/R group (Log2FC 2.7, Δβ = -0.14, p < 0.05). Based on these findings, we validated transcriptional and methylation changes of ASS1, PPARG, MYO1C and CDC42EP4 by RT-qPCR and bisulfite pyrosequencing using HL60 and MV4-11 AML cell lines in which DEC (IC50, HL60, 249 nM; MV4-11, 2090 nM) and VEN (IC50, HL60, 18 nM; MV4-11, 64 nM) produced additive or synergistic combination effect. Cells were treated by DEC and/or VEN under BM-derived mesenchymal stem cells (MSCs) co-culture conditions to mimic the BM microenvironment. DEC upregulated gene expression levels with TSS demethylation of PPARG, MYO1C and CDC42EP4 both in HL60 and MV4-11, and of ASS1 only in HL-60. VEN treatment alone or in combination with DEC did not induce significant changes in the expression of these tested genes. Of note, PPARG expression was further upregulated under MSCs co-culture condition without TSS methylation status change in AML cells. DEC treatment induced no significant changes of PPARG in MSC. These findings suggest that not only TSS demethylation but also interaction with BM stromal cells may be involved in PPARG upregulation associated with energy production in AML cells. Taken together, DEC-VEN treatment stimulates two key factors, energy metabolism and interaction with the BM microenvironment, which may reduce therapeutic efficacy by increasing gene expression of PPARG,MYO1C,ASS1, and CDC42EP4 through TSS demethylation and/or interaction with BM stromal cells. Experimentally, the consequences of overexpression / knockdown of these genes will be further validated.

Abstract A038: High-dimensional, multi-omics analyses of proteins, metabolites, transcripts, and genes enable biomarker discovery in early- and late-stage pancreatic cancer

Abstract Pancreatic cancer is the third leading cause of cancer-related deaths in the United States. Disease biomarkers quantified from blood-based assays may help reduce mortality by enabling early detection, treatment selection, or response and resistance assessment. PrognomiQ has developed a multi-omics assay and analysis platform that comprehensively profiles blood samples to detect proteins, metabolites, lipids, mRNA, miRNA, cfDNA fragments, and methylation at CpG sites. This platform can provide deep insights into the biology of pancreatic cancer and could enable the development of high sensitivity and specificity tests for the early detection of pancreatic cancer. We conducted a case-control study comprising 196 subjects: 92 with untreated pancreatic cancer and 104 matched controls without pancreatic cancer. For each subject, blood was collected in assay-specific tubes and processed to provide 7 different `omics readouts. cfDNA and mRNA were isolated from samples and assayed following standard NGS protocols. cfDNA fragments were processed to estimate fragment-length disorder and copy-number variation along with CpG site methylation. In addition, targeted and untargeted LCMS were used to detect and quantify proteins, metabolites, and lipids. After normalization, non-parametric univariate analyses of cases versus controls were performed to identify differentially abundant features on all available samples for each assay. Unsupervised learning was used to investigate the separation of subjects into groups based on disease status for the subset of 157 subjects for which complete data on all 7 readouts were available. We detected 2,812 proteins, 811 lipids, 373 metabolites, and 110,864 mRNA transcripts in all samples where data for each assay was available. Of these, 275 proteins, 232 lipids, 49 metabolites, and 3385 mRNA transcripts were significantly different (FWER &amp;lt; 0.05) in cases versus controls. From cfDNA data, we identified 35 non-contiguous genomic regions associated with fragment-length disorder, 557 with copy-number variation, and 5 with multiple, differentially methylated CpGs (FWER &amp;lt; 0.05) that aggregately span 307 protein-coding genes; of these, the overlap with the differentially expressed proteins included E-cadherin (tumor suppressor) and N-cadherin (involved in epithelial-to-mesenchymal transition). Statistically significant genes and proteins were found to be associated with processes including Wnt signaling, regulation of focal adhesion assembly, and actin cytoskeleton organization. Multi-omics, unsupervised learning showed separation of early- and late-stage cases and controls. High-dimensional bioinformatics analyses systematically decomposed each `omics data type into joint and orthogonal components associated with pancreatic cancer. Ongoing multivariate analyses, including supervised machine learning, will further elucidate the biology of pancreatic cancer development, and serve as the foundation for high-sensitivity blood tests for the early detection and monitoring of pancreatic cancer. Citation Format: Ehdieh Khaledian, Preethi Prasad, John Blume, Ghristine Bundalian, Connie Chao-Shern, Jinlyung Choi, Rea Cuaresma, Jared Deyarmin, Jun Heok Jang, Manoj Khadka, Thidar Khin, Yuya Kodama, Ajinkya Kokate, Joon-Yong Lee, Manway Liu, Nithya Mudaliar, Madhuvanthi Ramaiah, Saividya Ramaswamy, Peter Spiro, Kavya Swaminathan, Preston Williams, Mi Yang, James Yee, Brian Young, Robert Zawada, Susan Zhang, Chinmay Belthangady, Bruce Wilcox, Philip Ma. High-dimensional, multi-omics analyses of proteins, metabolites, transcripts, and genes enable biomarker discovery in early- and late-stage pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A038.

Multi-Omics Reveal Plasma Molecular Signatures of B-ALL Patients during CAR-T Cell Therapy

Background: The Chimeric antigen receptor (CAR) T cell therapy for hematological malignancies has revolutionized anticancer therapy. However, CAR-T cells can cause immunity-related adverse events, such as cytokine release syndrome and neurotoxicity; the efficacy of CAR-T cell therapy also varies substantially with different patients. Poor understanding of plasma proteomic and metabolomic features during chimeric antigen receptor (CAR) T cell therapy may affect the exploitation of therapeutic potential in the management of hematological malignancies. Methods: We longitudinally performed proteomic and metabolomic profiling of plasma from 20 patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) on CAR-T cell therapy. Blood samples were collected from B-ALL patients at different time points, including before fludarabine/cyclophosphamide lymphodepletion (Pre-FC), Day 0 (D0), Day 7 (D7), Day 14 (D14), Day 28 (D28), and Peak-IL-6 during CAR-T cell therapy. The plasma samples from 22 age- and gender-matched healthy controls (HC) were used as controls. Results: Compared with HC, 239, 217, 282, 260 and 261 proteins were markedly different in terms of expression level in these five groups (Pre-FC, D0, D7, D14, D28 versus HC), respectively (Figure 1). The pathways of differentially expressed proteins (DEPs) were highly enriched in processes implicated in actin binding, cadherin binding, regulation of ion transport, platelet aggregation and regulation of actin cytoskeleton, with actin binding obtaining the most significant change (Figure 1). 104, 139, 175, 223 and 87 metabolites were significantly different in these five groups, respectively (Figure 1). Interestingly, the alterations of plasma metabolites became more extensive on the 14th day after CAR-T cell infusion, indicating that the change of metabolites may lag behind that of proteins. Furthermore, CAR-T cell therapy reshaped plasma proteome and metabolome with discrete kinetics, as represented by upregulated acute-phase response and triglycerides metabolism and downregulated actin cytoskeleton organization, retrograde endocannabinoid signaling and glycosylphosphatidylinositol (GPI)-anchor biosynthesis during the therapy. We also identified sets of covarying molecules that were involved in acute inflammatory response, epithelial mesenchymal transition, glutathione metabolism and tryptophan metabolism in concert with the dynamics of circulating IL-6 in patients with cytokines storm. Subgroup analysis further revealed numerous prognostic biomarkers, such as proteins related to allograft rejection, which were down-regulated in patients who achieved stable complete remission. Conclusions: This study provided a dynamic landscape of plasma proteome and metabolome in B-ALL patients during CAR-T cell therapy and offered novel insights into CAR-T cell therapy-related toxicities and prognostic heterogeneity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Sec14-like-Lipid-Binding-4 Enables Hemoglobin Clearance By Liver Sinusoidal Endothelial Cells

Hemolysis of banked red blood cells (RBC storage lesion) contributes to lack of therapeutic benefit and higher mortality following transfusion therapy in patients. However, the molecular mechanism underlying storage lesion remains poorly understood. Using a multiple-ancestry genome-wide association analysis among blood donors, our group has recently identified Sec14-like-lipid-binding-4 (Sec14L4) protein to be associated with increased rate of hemolysis following blood storage. Although Sec14L4 is highly similar to yeast phosphatidylinositol transfer protein Sec14, which is known to be involved in the synthesis of golgi-derived transport vesicles, the exact function of Sec14L4 and how it promotes RBC storage lesions remains unknown. Our initial studies using fluorescence microscopy in our newly generated Sec14L4 null mice revealed significant enrichment of Sec14L4 in the live sinusoidal endothelial cells (LSECs), which positively correlated with elevated levels of iron in the liver. Scanning and transmission electron micrographs revealed impaired actin cytoskeleton organization in LSECs of Sec14L4 null mice, which was associated with upregulation of major GTPases RhoA, RhoB and Rac1 activity. The impairment of actin cytoskeletal organization in Sec14L4 null mice lead to impaired vesicle trafficking in LSECs and higher retention of RBCs in the liver sinusoids compared to control mice. These data are the first to demonstrate a previously unrecognized role for Sec14L4 protein in LSEC mediated endocytic trafficking by promoting actin polymerization. These findings also suggest that Sec14L4 may regulate RBC storage lesion through impaired hemoglobin clearance by LSECs secondary to cytoskeletal impairment.