Sustained Cell Proliferation Research Articles

The Role of Reprogrammed Glucose Metabolism in Cancer

Cancer cells reprogram their metabolism to meet biosynthetic needs and to adapt to various microenvironments. Accelerated glycolysis offers proliferative benefits for malignant cells by generating glycolytic products that move into branched pathways to synthesize proteins, fatty acids, nucleotides, and lipids. Notably, reprogrammed glucose metabolism and its associated events support the hallmark features of cancer such as sustained cell proliferation, hijacked apoptosis, invasion, metastasis, and angiogenesis. Overproduced enzymes involved in the committed steps of glycolysis (hexokinase, phosphofructokinase-1, and pyruvate kinase) are promising pharmacological targets for cancer therapeutics. In this review, we summarize the role of reprogrammed glucose metabolism in cancer cells and how it can be manipulated for anti-cancer strategies.

Autophagy Is Required to Sustain Increased Intestinal Cell Proliferation during Phenotypic Plasticity Changes in Honey Bee (Apis mellifera).

Tissue phenotypic plasticity facilitates rapid adaptation of organisms to biotic and/or abiotic pressure. The reproductive capacity of honey bee workers (Apis mellifera) is plastic and responsive to pheromones produced by broods and the queen. Egg laying workers (ELWs), which could reactivate their ovaries and lay haploid eggs upon queen lost, have been commonly discussed from many aspects. However, it remains unclear whether midgut homeostasis in ELWs is affected during plastic changes. Here, we found that the expression of nutrition- and autophagy-related genes was up-regulated in the midguts of ELWs, compared with that in nurse workers (NWs) by RNA-sequencing. Furthermore, the area and number of autophagosomes were increased, along with significantly increased cell death in the midguts of ELWs. Moreover, cell cycle progression in the midguts of ELWs was increased compared with that in NWs. Consistent with the up-regulation of nutrition-related genes, the body and midgut sizes, and the number of intestinal proliferation cells of larvae reared with royal jelly (RJ) obviously increased more than those reared without RJ in vitro. Finally, cell proliferation was dramatically suppressed in the midguts of ELWs when autophagy was inhibited. Altogether, our data suggested that autophagy was induced and required to sustain cell proliferation in ELWs' midguts, thereby revealing the critical role of autophagy played in the intestines during phenotypic plasticity changes.

Autophagy for secretory protein: Therapeutic targets in cancer.

Autophagy, a classical cellular degradative catabolic process, also involves a functionally discrete non-degradative role in eukaryotic cells. It imparts critical regulatory function on conventional and unconventional protein secretion (degradative and secretory autophagy with distinct lysosomal degradation and extracellular expulsion, respectively) pathways. The N-amino terminal leader sequence containing proteins follows a conventional secretion pathway, while the leader-less proteins opt for secretory autophagy. The secretory autophagic process ensembles core autophagy machinery proteins, specifically ULK1/2, Beclin 1, LC3, and GABARAP, in coordination with Golgi re-assembly and stacking proteins (GRASPs). The secretory omegasomes fuse with the plasma membrane for the expulsion of cytosolic cargos to the extracellular environment. Alternatively, the secretory omegasomes also fuse with multi-vesicular bodies (MVBs) and harmonize ESCRTs (Complex I; TSG101) and Rab GTPase for their release to extracellular space. Autophagy has been associated with the secretion of diverse proteins involved in cellular signaling, inflammation, and carcinogenesis. Secreted proteins play an essential role in cancer by sustaining cell proliferation, inhibiting apoptosis, enhancing angiogenesis and metastasis, immune cell regulation, modulation of cellular energy metabolism, and resistance to anticancer drugs. The complexity of autophagy regulation during tumorigenesis is dependent on protein secretion pathways. Autophagy-regulated TOR-autophagy spatial coupling compartment complex energizes enhanced secretion of pro-inflammatory cytokines and leaderless proteins such as HMGB1. In conclusion, the chapter reviews the role of autophagy in regulating conventional and unconventional protein secretion pathways and its possible role in cancer.

Quantification of Intracellular ATP Content in Ex Vivo GC B Cells.

The study of immunometabolism is an important and emerging field in immunology. B-cell activation upon antigen recognition induces profound metabolic changes in the cell, leading to an increase in ATP production to sustain cell proliferation and differentiation. Current methods available to determine the amount of ATP are time-consuming, require extensive sample processing, and need a large amount of starting material. We set up an easy follow-up protocol to determine the relative amount of ATP in living cells, combining cell surface staining with quinacrine. This acridine dye emits a green fluorescent signal in the presence of intracellular ATP. This protocol allows us to determine ATP in small populations of cells using flow cytometry, such as the germinal center.

Extracellular Matrix Secretion Mechanically Reinforces Interlocking Interfaces.

Drawing inspiration for biomaterials from biological systems has led to many biomedical innovations. One notable bioinspired device, Velcro, consists of two substrates with interlocking ability. Generating reversibly interlocking biomaterials is an area of investigation, as such devices can allow for modular tissue engineering, reversibly interlocking biomaterial interfaces, or friction-based coupling devices. Here, a biaxially interlocking interface generated using electrostatic flocking is reported. Two electrostatically flocked substrates are mechanically and reversibly interlocked with the ability to resist shearing and compression forces. An initial high-throughput screen of polyamide flock fibers with varying diameters and fiber lengths is conducted to elucidate the roles of different fiber parameters on scaffold mechanical properties. After determining the most desirable parameters via weight scoring, polylactic acid (PLA) fibers are used to emulate the ideal scaffold for in vitro use. PLA flocked scaffolds are populated with osteoblasts and interlocked. Interlocked flocked scaffolds improved cell survivorship under mechanical compression and sustained cell viability and proliferation. Additionally, the compression and shearing resistance of cell-seeded interlocking interfaces increased with increasing extracellular matrix deposition. The introduction of extracellular matrix-reinforced interlocking interfaces may serve as binders for modular tissue engineering, act as scaffolds for engineering tissue interfaces, or enable friction-based couplers for biomedical applications.

ERBB2 mutation is associated with sustained tumor cell proliferation after short-term preoperative endocrine therapy in early lobular breast cancer

Invasive lobular breast cancer (ILC) is a special breast cancer (BC) subtype and is mostly hormone receptor (HR)-positive and ERBB2 non-amplified. Endocrine therapy restrains tumor proliferation and is the mainstay of lobular BC treatment. Mutation of ERBB2 has been associated with recurrent ILC. However, it is unknown whether ERBB2 mutation impacts on the otherwise exquisite responsiveness of early ILC to endocrine therapy. We have recently profiled n = 622 HR-positive early BCs from the ADAPT trial for mutations in candidate genes involved in endocrine resistance, including ERBB2. All patients were treated with short-term preoperative endocrine therapy (pET, tamoxifen or aromatase inhibitors) before tumor resection. Tumor proliferation after endocrine therapy (post-pET Ki67 index) was determined prospectively by standardized central pathology assessment supported by computer-assisted image analysis. Sustained or suppressed proliferation were defined as post-pET Ki67 ≥10% or <10%. Here, we report a subgroup analysis pertaining to ILCs in this cohort. ILCs accounted for 179/622 (28.8%) cases. ILCs were enriched in mutations in CDH1 (124/179, 69.3%, P < 0.0001) and ERBB2 (14/179, 7.8%, P < 0.0001), but showed fewer mutations in TP53 (7/179, 3.9%, P = 0.0048) and GATA3 (11/179, 6.1%, P < 0.0001). Considering all BCs irrespective of subtypes, ERBB2 mutation was not associated with proliferation. In ILCs, however, ERBB2 mutations were 3.5-fold more common in cases with sustained post-pET proliferation compared to cases with suppressed post-pET proliferation (10/75, 13.3% versus 4/104, 3.8%, P = 0.0248). Moreover, ERBB2 mutation was associated with high Oncotype DX recurrence scores (P = 0.0087). In summary, our findings support that ERBB2 mutation influences endocrine responsiveness in early lobular BC.

Single-Cell Sequencing Identifies Master Regulators Affected by Panobinostat in Neuroblastoma Cells.

The molecular mechanisms and gene regulatory networks sustaining cell proliferation in neuroblastoma (NBL) cells are still not fully understood. In this tumor context, it has been proposed that anti-proliferative drugs, such as the pan-HDAC inhibitor panobinostat, could be tested to mitigate tumor progression. Here, we set out to investigate the effects of panobinostat treatment at the unprecedented resolution offered by single-cell sequencing. We identified a global senescence signature paired with reduction in proliferation in treated Kelly cells and more isolated transcriptional responses compatible with early neuronal differentiation. Using master regulator analysis, we identified BAZ1A, HCFC1, MAZ, and ZNF146 as the transcriptional regulators most significantly repressed by panobinostat. Experimental silencing of these transcription factors (TFs) confirmed their role in sustaining NBL cell proliferation in vitro.

CSIG-37. MERLIN S13 DEPHOSPHORYLATION DRIVES MENINGIOMA WNT SIGNALLING AND CELL PROLIFERATION

Abstract How Merlin-intact meningiomas arise in the absence of NF2/Merlin inactivation is incompletely understood. Here, we integrate single-cell RNA sequencing of 86,000 cells from meningioma xenografts with APEX2 proteomic proximity-labelling mass spectrometry and functional biochemical approaches to discover Merlin Serine 13 (S13) dephosphorylation drives meningioma Wnt signalling and cell proliferation. Cell biology, molecular biology, and biochemical techniques were used to validate Merlin functions in meningioma cells or xenografts using wildtype Merlin constructs or Merlin constructs encoding S13A, phosphomimetic S13D, or cancer-associated missense substitutions (L46R, A211D). Single-cell RNA sequencing of meningioma xenografts showed Merlin rescue activated the Wnt pathway in Merlin-deficient meningiomas. Proteomic proximity-labelling mass spectrometry revealed b-catenin, PKC, and PP1A interactions with wildtype Merlin, but not with Merlin L46R or A211D. b-catenin does not interact with other FERM family members, and Merlin contains a unique N-terminal domain (NTD) with a PKC phosphorylation motif overlapping with a PP1A dephosphorylation motif at S13. Thus, we hypothesized the Merlin S13, PKC, and PP1A may be important for Wnt signalling in Merlin-intact meningiomas. In support of this hypothesis, over-expression of wildtype Merlin or Merlin S13A but not Merlin DNTD, S13D, L46R, A211D, or other FERM family members drove meningioma Wnt signalling and sustained meningioma cell proliferation in vivo. Moreover, b-catenin was detected in proximity to Merlin S13D but not Merlin S13A in meningioma cells. Meningioma cell fractionation and immunofluorescence showed Merlin S13D over-expression stabilized b-catenin at the plasma membrane and inhibited Wnt signalling. Phospho-proteomic mass spectrometry and custom phospho-specific antibodies integrated with shRNA or siRNA gene suppression demonstrated PKC phosphorylated Merlin S13, but meningioma Wnt pathway activation induced PP1A to dephosphorylate Merlin S13 and drive cell proliferation. In summary, Merlin S13 dephosphorylation drives meningioma Wnt signalling and cell proliferation. These data reveal a novel tumor-promoting function of NF2/Merlin in Merlin-intact meningiomas.

Liver regeneration after partial hepatectomy is improved in the absence of aryl hydrocarbon receptor

The liver is among the few organs having the ability to self-regenerate in response to a severe damage compromising its functionality. The Aryl hydrocarbon receptor (Ahr) is a transcription factor relevant for the detoxification of xenobiotics but also largely important for liver development and homeostasis. Hence, liver cell differentiation is developmentally modulated by Ahr through the controlled expression of pluripotency and stemness-inducing genes. Here, 2/3 partial hepatectomy (PH) was used as a clinically relevant approach to induce liver regeneration in Ahr-expressing (Ahr+/+) and Ahr-null (Ahr−/−) mice. Ahr expression and activity were early induced after 2/3 PH to be gradually downmodulated latter during regeneration. Ahr−/− mice triggered liver regeneration much faster than AhR+/+ animals, although both reached full regeneration at the latest times. At initial stages after PHx, earlier regenerating Ahr−/− livers had upregulation of cell proliferation markers and increased activation of signalling pathways related to stemness such as Hippo-YAP and Wnt/β-catenin, concomitantly with the induction of pro-inflammatory cytokines TNFa, IL6 and p65. These phenotypes, together with the improved metabolic adaptation of Ahr−/− mice after PHx and their induced sustained cell proliferation, could likely result from the expansion of undifferentiated stem cells residing in the liver expressing OCT4, SOX2, KLF4 and NANOG. We propose that Ahr needs to be induced early during regeneration to fine-tune liver regrowth to physiological values. Since Ahr deficiency did not result in liver overgrowth, its transient pharmacological inhibition could serve to improve liver regeneration in hepatectomized and transplanted patients and in those exposed to damaging liver toxins and carcinogens.

Design and optimization of oestrogen receptor PROTACs based on 4-hydroxytamoxifen

In the last four decades, treatment of oestrogen receptor positive (ER+) breast cancer (BCa), has focused on targeting the estrogenic receptor signaling pathway. This signaling function is pivotal to sustain cell proliferation. Tamoxifen, a competitive inhibitor of oestrogen, has played a major role in therapeutics. However, primary and acquired resistance to hormone blockade occurs in a large subset of these cancers, and new approaches are urgently needed. Aromatase inhibitors and receptor degraders were approved and alternatively used. Yet, resistance appears in the metastatic setting. Here we report the design and synthesis of a series of proteolysis targeting chimeras (PROTACs) that induce the degradation of estrogen receptor alpha in breast cancer MCF-7 (ER+) cells at nanomolar concentration. Using a warhead based on 4-hydroxytamoxifen, bifunctional degraders recruiting either cereblon or the Von Hippel Lindau E3 ligases were synthesized. Our efforts resulted in the discovery of TVHL-1, a potent ERα degrader (DC50: 4.5 nM) that we envisage as a useful tool for biological study and a platform for potential therapeutics.

Increased Stiffness Downregulates Focal Adhesion Kinase Expression in Pancreatic Cancer Cells Cultured in 3D Self-Assembling Peptide Scaffolds.

The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that participates in integrin-mediated signal transduction and contributes to different biological processes, such as cell migration, survival, proliferation and angiogenesis. Moreover, FAK can be activated by autophosphorylation at position Y397 and trigger different signaling pathways in response to increased extracellular matrix stiffness. In addition, FAK is overexpressed and/or hyperactivated in many epithelial cancers, and its expression correlates with tumor malignancy and invasion potential. One of the characteristics of solid tumors is an over deposition of ECM components, which generates a stiff microenvironment that promotes, among other features, sustained cell proliferation and survival. Researchers are, therefore, increasingly developing cell culture models to mimic the increased stiffness associated with these kinds of tumors. In the present work, we have developed a new 3D in vitro model to study the effect of matrix stiffness in pancreatic ductal adenocarcinoma (PDAC) cells as this kind of tumor is characterized by a desmoplastic stroma and an increased stiffness compared to its normal counterpart. For that, we have used a synthetic self-assembling peptide nanofiber matrix, RAD16-I, which does not suffer a significant degradation in vitro, thus allowing to maintain the same local stiffness along culture time. We show that increased matrix stiffness in synthetic 3D RAD16-I gels, but not in collagen type I scaffolds, promotes FAK downregulation at a protein level in all the cell lines analyzed. Moreover, even though it has classically been described that stiff 3D matrices promote an increase in pFAKY397/FAK proteins, we found that this ratio in soft and stiff RAD16-I gels is cell-type-dependent. This study highlights how cell response to increased matrix stiffness greatly depends on the nature of the matrix used for 3D culture.

Synthetic antioxidants from a natural source can overtake the oncogenic stress management system and activate thestress‑sensitized death of KSHV‑infected cancer cells.

Synthetic and modified natural derivatives are reported as potential bioactive compounds and are being used therapeutically against various diseases in a wide-spread manner nowadays. Cancerous cells exhibit high levels of reactive oxygen species (ROS) internally, and thus successfully manage to sustain themselves and proliferate via antioxidative mechanisms that maintain a redox balance. On this note, various antioxidants are applied as anticancer compounds, which strategically affects the ongoing oncogenic stress management system in both a pro- and antioxidative manner, resulting in cancer restriction, as well as sustaining cell proliferation via antioxidative mechanisms that promote cancer progression. Alike non-viral cancers, viral cancers exhibit varying levels of ROS during different stages of cancer progression. Hence, successful stress balance should be addressed, depending on the cancer cell stress response during the therapeutic management. The application of antioxidants is crucial and needs to be carefully designed in such cases; the respective underlying mechanisms are less understood. The role of antioxidants controlling the varied levels of stress response at different stages of Kaposi's sarcoma-associated herpes virus malignancy have not been fully reported. Therefore, the present study aimed to analyze the activity of certain antioxidants in KSHV-infected oncogenic cells. For this purpose, two naturally derived flavonoid-based antioxidants (theaflavin and novel curcumin derivatives) were selected and tested in different KSHV-infected cell lines. The findings presented herein demonstrate that these compounds can successfully induce the death of different KSHV-positive cells and can restrict the growth of KSHV-infected cell lines restricting viral reactivation by counteracting the oncogenic stress management system.

Taming of Singlet Oxygen: Towards Artificial Oxygen Carriers Based on 1,4-Dialkylnaphthalenes.

Naphthalene endoperoxides are known as convenient sources of singlet oxygen (O2 , 1 Δg ), which is the major product of endoperoxide cycloreversion reaction. However, their potential as carriers of ground-state molecular oxygen (O2 , 3 Σg ) similar to artificial oxygen carriers remains largely unexplored. This is due to the extreme reactivity and cytotoxic effects of the released singlet oxygen. We now report that a compound with a bimodular design, which incorporates an endoperoxide and an efficient physical quencher of singlet oxygen, 1,4-diazabicyclo[2.2.2]octane (DABCO), produces exclusively ground-state molecular oxygen. This result, coupled with the fact that oxygen release rates from endoperoxides are highly amenable to fine-tuning in a very broad range, and open to targeting by ligand attachment, raises the potential of these compounds far above any comparable chemical, or even biochemical sources. In cell culture experiments, we showed that the addition of the endoperoxide-quencher conjugate can enhance and sustain cell proliferation.

The potential implications of estrogenic and antioxidant-dependent activities of high doses of methyl paraben on MCF7 breast cancer cells.

Methyl paraben (MP) is an endocrine-disrupting compound that possesses estrogenic properties and contributes to an aberrant burden of estrogen signaling in the human breast and subsequently increasing the risks for the development of breast cancer. The exact exposure, as well as the safe concentrations, are variable among daily products. The present study addresses the effects of exposure to escalated concentrations of MP on the proliferation of MCF-7 breast cancer cells in addition to exploring its other mechanisms of action. The study involved exposure of cultured MCF-7 breast cancer cells to seven MP concentrations, ranging from 40 to 800 µM for 5 days. Cell viability, apoptosis, and proliferation were respectively assessed using crystal violet test, flow cytometric analysis, and quantitative real-time polymerase chain reaction for Ki-67 expression. The estradiol (E2) secretion and oxidative stress were also assessed and analyzed in correlation to MP's proliferation and cytotoxicity potentials. The results showed that the maximum proliferative concentration of MP was 800 µM. At a concentration of 40 μM and higher, MP induced increased expression of Ki-67, denoting enhanced proliferation of the cells in monolayer culture. A positive correlation between the detrimental oxidative stress effect of MP's tested concentrations, cell proliferation, and viability was demonstrated (p < 0.05). Our results indicated that MP at high doses induced sustained cell proliferation due to E2 secretion as well as its antioxidant activity. Accordingly, it was concluded that high and unpredicted exposure to MP might carry a carcinogenic hazard on estrogen receptor-positive breast cancer cells.

Degraders: The Ultimate Weapon Against Amplified Driver Kinases in Cancer.

Amplification of pro-oncogenic kinases is a common genetic alteration driving tumorigenic phenotypes. Cancer cells rely on the amplified kinases to sustain cell proliferation, survival, and growth, presenting an opportunity to develop therapies targeting the amplified kinases. Utilizing small molecule catalytic inhibitors as therapies to target amplified kinases is plagued by de novo resistance driven by increased expression of the target, and amplified kinases can drive tumorigenic phenotypes independent of catalytic activity. Here, we discuss the emergence of proteolysis-targeting chimeras that provide an opportunity to target these oncogenic drivers effectively.SIGNIFICANCE STATEMENTProtein kinases contribute to tumorigenesis through catalytic and noncatalytic mechanisms, and kinase gene amplifications are well described mechanisms of resistance to small molecule catalytic inhibitors. Repurposing catalytic inhibitors for the development of protein degraders will offer improved clinical benefits by targeting noncatalytic functions of kinases that promote tumorigenesis and overcoming resistance due to amplification.

FGF10 protects against particulate matter (PM)-induced lung injury via regulation of endoplasmic reticulum stress

Exposure of the lungs to particulate matter (PM) leads to the development of respiratory disease and involves mechanisms such as oxydative stress, mitochondrial dysfunction and endoplasmic reticulum (ER) stress. However, there are no effective therapies to treat PM-induced lung diseases. Fibroblast growth factor 10 (FGF10) is a multifunctional growth factor mediating mesenchymal-to-epithelial signaling and displaying a significant therapeutic potential following injury. The present research aims to investigate the regulatory mechanism of FGF10 on ER stress in PM-induced lung injury. PM-induced lung injury leads to peribronchial wall thickening and marked infiltration of inflammatory cells which is associated with increased secretion of inflammatory cytokines. The results show that FGF10 treatment attenuates PM-induced lung injury in vivo and reversed ER stress protein GRP78 and CHOP levels. Moreover, comparison of human bronchial epithelial cells cultured with PM and FGF10 vs PM alone shows sustained cell proliferation and restrained secretion of inflammatory cytokines supporting FGF10′s protective role. Significantly, both ERK1/2 and PI3K/AKT inhibitors largely abolished the impact of FGF10 on PM-induced ER stress. Taken together, both in vivo and in vitro experiments showed that FGF10, via the activation of ERK1/2 and PI3K/AKT signaling, protects against PM-induced lung injury through the regulation of ER stress. Therefore, FGF10 represents a potential therapy for PM-induced lung injury.

Natural origin bilayer pullulan-PHBV scaffold for wound healing applications

Skin tissue loss that occurs by injury and diseases can turn into chronic wounds as a result of complications alongside infection. Chronic wounds fail to heal by themselves and need advanced treatments like engineered wound dressings and regenerative scaffolds. In this study, a novel, natural origin, bilayer electrospun scaffold was produced from pullulan (PUL) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) biopolymers. PHBV production by Cupriavidus necator bacterial strain was optimized and produced polymer was characterized. Characteristic peaks and bands of PHBV were observed by H-NMR and FTIR analyses. Valerate mol percent of produced PHBV copolymer was determined by H-NMR. Average molecular weight of the polymer was determined by SLS technique and crystallinity of PHBV was calculated from DSC curve. Bilayer scaffold was produced by electrospinning of hydrophilic PUL fibrous membrane onto wet-electrospun hydrophobic PHBV 3D fibrous mat. Bilayer scaffold was designed to involve regenerative and barrier fibrous layers. Nano fibrous PUL membrane with smaller pore size was efficient as a barrier against bacterial transmission while enabling optimum oxygen and water vapor transmission. Water retention and degradation properties were found to be optimum for a skin tissue scaffold. In vitro studies showed that PUL membrane sustained L929 cell proliferation while preventing cells from migrating inside the barrier phase while PHBV layer supported cell viability, proliferation, and migration, creating a regenerative 3D structure. Results showed that, novel natural origin PUL/PHBV bilayer scaffold is a promising candidate for wound healing applications.

In-situ monitoring of defects in extrusion-based bioprinting processes using visible light imaging

Tissue engineering techniques are central for the development of biomedical scaffolds, which are primarily employed in the biofabrication of various artificial human tissue and organ models. Bioprinting is a new technique of creating tissue constructs that can sustain cell proliferation. The development of printing techniques proceeds together with the development of the biomaterials to be printed, which is why studying the printability of these specific biomaterials must be explored. An appropriate hydrogel used as bioink should have numerous rheological, mechanical, and biological properties for producing appropriate tissue constructs. However, reaching the right trade-off between a desirable bioactivity and high printability is challenging, and despite numerous optimization studies for different materials, printing defects often occur during printing. Herein, methods are proposed to automatically identify these drifting processes in commonly used geometries and how they affected subsequent layers, as well as printing defects within each layer. Several structures were printed with standard commercial bioink as proof of concept. The constructs were analyzed using optical images from a coaxial camera. The images were then digitally processed to get geometrical data from which patterns of defectology to be monitored were derived. This automation should decrease the time in post-processing characterization of constructs and should provide a standardized tool to compare different bioinks.

Regulation of Liver Regeneration by Hepatocyte O-GlcNAcylation in Mice.

Background & AimsThe liver has a unique capacity to regenerate after injury in a highly orchestrated and regulated manner. Here, we report that O-GlcNAcylation, an intracellular post-translational modification regulated by 2 enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a critical termination signal for liver regeneration following partial hepatectomy (PHX).MethodsWe studied liver regeneration after PHX on hepatocyte specific OGT and OGA knockout mice (OGT-KO and OGA-KO), which caused a significant decrease (OGT-KO) and increase (OGA-KO) in hepatic O-GlcNAcylation, respectively.ResultsOGA-KO mice had normal regeneration, but the OGT-KO mice exhibited substantial defects in termination of liver regeneration with increased liver injury, sustained cell proliferation resulting in significant hepatomegaly, hepatic dysplasia, and appearance of small nodules at 28 days after PHX. This was accompanied by a sustained increase in expression of cyclins along with significant induction in pro-inflammatory and pro-fibrotic gene expression in the OGT-KO livers. RNA-sequencing studies revealed inactivation of hepatocyte nuclear 4 alpha (HNF4α), the master regulator of hepatic differentiation and a known termination signal, in OGT-KO mice at 28 days after PHX, which was confirmed by both Western blot and immunohistochemistry analysis. Furthermore, a significant decrease in HNFα target genes was observed in OGT-KO mice, indicating a lack of hepatocyte differentiation following decreased hepatic O-GlcNAcylation. Immunoprecipitation experiments revealed HNF4α is O-GlcNAcylated in normal differentiated hepatocytes.ConclusionsThese studies show that O-GlcNAcylation plays a critical role in the termination of liver regeneration via regulation of HNF4α in hepatocytes.

A Multi-Omics Approach for Evaluating the Impact of Cytokines and Donor Source on NK Cell Expansion

IntroductionAs the field of cancer immunotherapy progresses, natural killer (NK) cells have become an ideal candidate for targeting multiple types of cancer. These cells are able to selectively target virally infected cells and tumor cells without damaging the healthy cells of the immune system making them an ideal candidate for treatment in AML, CML, MM, Neuroblastoma, Breast Cancer, and Renal Cell Carcinoma. While NK cells have shown their potential as immunotherapy agents, one of the biggest challenges includes expanding and harvesting enough cells for multiple transfusions when there are limited numbers of starting cells. Our group developed an NK cell expansion platform utilizing membrane bound-IL21 (mbIL-21), which allows for sustained proliferation in order to generate high numbers of cells. Continued research supported that mbIL-21 enables sustained NK cell proliferation compared to mbIL-15 due to mbIL-21 activation through STAT3 signaling. Recent shifts to umbilical cord blood have led to research using cord blood (CB) derived NK cells. However, this presents a challenge as a viable clinical option as others have shown that the expansion of these CB-derived NK cells have been shown to produce less potent NK cells. Here, we will use feeder cells expressing either mbIL15 or mbIL21 to expand NK cells (as is currently done in clinical trials), to deeply compare and contrast the impact of these two cytokines on NK cell biology and simultaneously compare and contrast the use of peripheral-derived NK cells and CB-derived NK cells.MethodsNK cells were obtained from healthy donor leukopacks (n =4) and isolated using RosetteSep human NK cell enrichment cocktail. These samples were stimulated with either mbIL-21 or mbIL-15 irradiated feeder cells (IFC) and cultured in AIM-V media supplemented with 100 I/U of IL-2 and immune cell serum replacement (ICSR). Cord Blood NK cells were obtained from OrganaBio (n=4) and stimulated with mbIL-21 IFC . The cells were cultured in AIM-V media supplemented with 100 I/U of IL-2 and ICSR (complete media). All NK cell expansions were stimulated at day 0 and day 7, as previously described, and cell cultures were assessed every 2-3 days keep cells at an optimal concentration by supplementing with complete media. Cells were collected at day 14 to capture a picture before mbIL-15 expanded cells became senescent and were immediately used for cytokine production assays, RNA collected, ATAC-seq collection, and CyTOF fixation. The remaining cells were frozen for cytotoxicity assays, mitochondrial function assays, and degranulation assays. For mitochondrial function and cytotoxicity assays, cells were thawed and cultured in complete media for two days before use.ResultsOverall, the peripheral blood NK cells expanded with mbIL21 showed an increase in expansion at day 14 when compared to those expanded with mbIL15. NK cells expanded with mbIL21 demonstrated a more consistent cytotoxicity profile against CHLA-136, a neuroblastoma cell line, compared to mbIL21 expanded NK cells when cultured at a 0.5:1 ratio (E:T). While the results were not statistically significant at 14 days, previous data demonstrates that we see increased changes at 21 days. Of note, we had an overall more reproducible serial killing ability in the IL21 expanded samples compared to the variability observed in IL15-expanded samples. We saw a significant difference (p < 0.0001) in peripheral blood NK cells expanded with mbIL21 compared to cb-NK cells expanded with mbIL21 with a 0.5:1 ratio (E:T) against CHLA136. mbIL-21 expanded peripheral blood NK cells had a higher maximum respiration capacity than those expanded with mbIL-15 when measured under mitochondrial stress via seahorse assay (p= 0.016).ConclusionsOur data showed that the cells began to show a different phenotypic profile at day 14 depending on the type of NK cell and the type of cytokine used for stimulation. By day 14 data supported that mbIL-21 expanded cells can better survive under low glucose conditions such as tumor microenvironments than mbIL15 expanded cells. The cytotoxicity data demonstrated that peripheral derived NK cells have improved serial killing ability than the cb-NK cells and we will continue to test their ability against additional tumor cell lines that have various HLA-typing. To further elucidate these differences, we will use our RNA-seq, ATAC-seq, and CyTOF data to understand what is causing the different profiles. [Display omitted] DisclosuresLee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company.