Loss Of Proliferative Capacity Research Articles

Clinical validation of C12FDG as a marker associated with senescence and osteoarthritic phenotypes.

Chronic conditions associated with aging have proven difficult to prevent or treat. Senescence is a cell fate defined by loss of proliferative capacity and the development of a pro-inflammatory senescence-associated secretory phenotype comprised of cytokines/chemokines, proteases, and other factors that promotes age-related diseases. Specifically, an increase in senescent peripheral blood mononuclear cells (PBMCs), including T cells, is associated with conditions like frailty, rheumatoid arthritis, and bone loss. However, it is unknown if the percentage of senescent PBMCs associated with age-associated orthopedic decline could be used for potential diagnostic or prognostic use in orthopedics. Here, we report senescent cell detection using the fluorescent compound C12FDG to quantify PBMCs senescence across a large cohort of healthy and osteoarthritic patients. There is an increase in the percent of circulating C12FDG+ PBMCs that is commensurate with increases in age and senescence-related serum biomarkers. Interestingly, C12FDG+ PBMCs and T cells also were found to be elevated in patients with mild to moderate osteoarthritis, a progressive joint disease that is strongly associated with inflammation. The percent of C12FDG+ PBMCs and age-related serum biomarkers were decreased in a small subgroup of study participants taking the senolytic drug fisetin. These results demonstrate quantifiable measurements in a large group of participants that could create a composite score of healthy aging sensitive enough to detect changes following senolytic therapy and may predict age-related orthopedic decline. Detection of peripheral senescence in PBMCs and subsets using C12FDG may be clinically useful for quantifying cellular senescence and determining how and if it plays a pathological role in osteoarthritic progression.

A kinase-dead natural polymorphism in the canine Tnni3k gene.

Most mammalian cardiomyocytes become polyploid in the neonatal period, concurrent with their loss of proliferative capacity. In mice, natural or engineered mutation of the cardiomyocyte-specific kinase gene Tnni3k causes a higher level of diploid CMs and a higher capacity to support proliferation after adult injury. Here, we identified a polymorphism in the canine Tnni3k gene that is particularly common in the West Highland White Terrier breed, and show that this variant eliminates Tnni3k kinase activity. Thus, in several species, natural Tnni3k polymorphisms exist that are predicted to contribute to variation in diploid CM level and heart regenerative ability.

Implications of Senescent T Cells for Cancer Immunotherapy.

T-cell senescence is thought to result from the age-related loss of the ability to mount effective responses to pathogens and tumor cells. In addition to aging, T-cell senescence is caused by repeated antigenic stimulation and chronic inflammation. Moreover, we demonstrated that T-cell senescence was induced by treatment with DNA-damaging chemotherapeutic agents. The characteristics of therapy-induced senescent T (TIS-T) cells and general senescent T cells are largely similar. Senescent T cells demonstrate an increase in the senescence-associated beta-galactosidase-positive population, cell cycle arrest, secretion of senescence-associated secretory phenotypic factors, and metabolic reprogramming. Furthermore, senescent T cells downregulate the expression of the co-stimulatory molecules CD27 and CD28 and upregulate natural killer cell-related molecules. Moreover, TIS-T cells showed increased PD-1 expression. However, the loss of proliferative capacity and decreased expression of co-stimulatory molecules associated with T-cell senescence cause a decrease in T-cell immunocompetence. In this review, we discuss the characteristics of senescent T-cells, including therapy-induced senescent T cells.

Aging and Metabolic Reprogramming of Adipose-Derived Stem Cells Affect Molecular Mechanisms Related to Cardiovascular Diseases.

We performed a systematic search of the PubMed database for English-language articles related to the function of adipose-derived stem cells in the pathogenesis of cardiovascular diseases. In preclinical models, adipose-derived stem cells protected arteries and the heart from oxidative stress and inflammation and preserved angiogenesis. However, clinical trials did not reiterate successful treatments with these cells in preclinical models. The low success in patients may be due to aging and metabolic reprogramming associated with the loss of proliferation capacity and increased senescence of stem cells, loss of mitochondrial function, increased oxidative stress and inflammation, and adipogenesis with increased lipid deposition associated with the low potential to induce endothelial cell function and angiogenesis, cardiomyocyte survival, and restore heart function. Then, we identify noncoding RNAs that may be mechanistically related to these dysfunctions of human adipose-derived stem cells. In particular, a decrease in let-7, miR-17-92, miR-21, miR-145, and miR-221 led to the loss of their function with obesity, type 2 diabetes, oxidative stress, and inflammation. An increase in miR-34a, miR-486-5p, and mir-24-3p contributed to the loss of function, with a noteworthy increase in miR-34a with age. In contrast, miR-146a and miR-210 may protect stem cells. However, a systematic analysis of other noncoding RNAs in human adipose-derived stem cells is warranted. Overall, this review gives insight into modes to improve the functionality of human adipose-derived stem cells.

Development of a DNA damage-induced senescence model in osteoarthritic chondrocytes.

Senescent cells (SnCs) have been described to accumulate in osteoarthritis (OA) joint tissues in response to injury, thereby participating in OA development and progression. However, clinical therapeutic approaches targeting SnCs using senolysis, although promising in preclinical OA models, have not yet proven their efficacy in patients with knee OA. This pitfall may be due to the lack of understanding of the mechanisms underlying chondrocyte senescence. Therefore, our study aimed to generate models of chondrocyte senescence. This study used etoposide, to induce DNA damage-related senescence or chronic exposure to IL-1β to entail inflammation-related senescence in human OA chondrocytes. Several hallmarks of cellular senescence, such as cell cycle arrest, expression of cyclin-dependent kinase inhibitors, DNA damages, and senescence-associated secretory profile were evaluated. Chronic exposure to IL-1β induces only partial expression of senescence markers and does not allow us to conclude on its ability to induce senescence in chondrocytes. On the other hand, etoposide treatment reliably induces DNA damage-related senescence in human articular chondrocytes evidenced by loss of proliferative capacity, DNA damage accumulation, and expression of some SASP components. Etoposide-induced senescence model may help investigate the initiation of cellular senescence in chondrocytes, and provide a useful model to develop therapeutic approaches to target senescence in OA.

A superoxide-driven redox state promotes geroconversion and resistance to senolysis in replication-stress associated senescence

Using S-phase synchronized RPE1-hTERT cells exposed to the DNA damaging agent, methyl methanesulfonate, we show the existence of a redox state associated with replication stress-induced senescence termed senescence-associated redox state (SA-redox state). SA-redox state is characterized by its reactivity with superoxide-sensing fluorescent probes such as dihydroethidine, lucigenin and mitosox and peroxynitrite or hydroxyl radical sensing probe hydroxyphenyl fluorescein (HPF) but not the hydrogen peroxide (H2O2) reactive fluorescent probe CM-H2DCFDA. Measurement of GSH and GSSH also reveals that SA-redox state mitigates the level of total GSH rather than oxidizes GSH to GSSG. Moreover, supporting the role of superoxide (O2.-) in the SA-redox state, we show that incubation of senescent RPE1-hTERT cells with the O2.- scavenger, Tiron, decreases the reactivity of SA-redox state with the oxidants’ reactive probes lucigenin and HPF while the H2O2 antioxidant N-acetyl cysteine has no effect. SA-redox state does not participate in the loss of proliferative capacity, G2/M cell cycle arrest or the increase in SA-β-Gal activity. However, SA-redox state is associated with the activation of NF-κB, dictates the profile of the Senescence Associated Secretory Phenotype, increases TFEB protein level, promotes geroconversion evidenced by increased phosphorylation of S6K and S6 proteins, and influences senescent cells response to senolysis. Furthermore, we provide evidence for crosstalk between SA redox state, p53 and p21. While p53 mitigates the establishment of SA-redox state, p21 is critical for the sustained reinforcement of the SA-redox state involved in geroconversion and resistance to senolysis.

Abstract 1428: Luminescent immunoassays for homogeneous determination of cell proliferation and senescence

Abstract We have developed homogeneous, luminescent immunoassays for simple and rapid determination of Ki-67 and uPAR levels in 96-well format, thereby enabling efficient and quantitative assessment of proliferation and senescence in mammalian cell culture. Assessment of proliferative capacity is of key importance in numerous research areas, including cancer and various proliferative disorders, assessment of cell fitness (e.g., T cell fitness, stem cell characterization), and evaluation of test compound or drug effects. Cell senescence, characterized in part by loss of proliferative capacity, is an altered, proinflammatory state of cells induced in response to developmental ques, aging, disease, or acute stressors typically involving DNA damage. Ki-67 is a nuclear protein widely used as a marker of proliferation due to its pervasive expression in cycling cells but absence from non-dividing (G0) cells, whether quiescent, senescent, or terminally differentiated. In contrast, urokinase plasminogen activator receptor (uPAR) is a cell-surface protein whose expression was recently reported to increase significantly with the induction of senescence. Of note, commonly used assays of Ki-67 and uPAR levels typically rely upon non-homogeneous methods, including imaging, flow cytometry, or western blotting. Application of Lumit™ technology enabled our development of no-transfer, no-wash immunoassays for Ki-67 (lytic) and uPAR (nonlytic) determinations directly in cell wells. Treatment of human CD8+ T cells with CD3/CD28 T cell activator produced more than a 10-fold upregulation of Ki-67 levels, consistent with induction of T cell proliferation. In contrast, treatment of HCT116 colorectal cancer cells for 48 h with the antiproliferative compounds doxorubicin (100 nM) and nutlin-3A (10 μM) produced 65% and 90% reductions, respectively, in Ki-67 levels normalized to a measure of viable cell number, consistent with their relative antiproliferative activities. Dose-dependent treatment of HCT116 cells with nutlin-3A for 24 and 48 h produced 69% and 93% reduction, respectively, in normalized Ki-67 levels with a potency of ~2.5 μM. Treatment of HCT116 and A549 cancer cell lines for 72 h with 10-20 μM nutlin-3A, in addition to causing profound reduction in Ki-67 levels and loss of proliferative capacity, produced 2.3- to 4-fold increases in cell-surface uPAR levels, reflective of the emergence of the senescence phenotype. These luminescent, homogeneous Ki-67 and uPAR immunoassays provide an efficient means to quantitatively assess changes in cell proliferation and senescence in cell culture models for basic research and drug discovery applications. Citation Format: Dan F. Lazar, Kevin R. Kupcho, Andrew L. Niles, James J. Cali. Luminescent immunoassays for homogeneous determination of cell proliferation and senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1428.

Cardiomyocyte ploidy is dynamic during postnatal development and varies across genetic backgrounds.

Somatic polyploidization, an adaptation by which cells increase their DNA content to support growth, is observed in many cell types, including cardiomyocytes. Although polyploidization is believed to be beneficial, progression to a polyploid state is often accompanied by loss of proliferative capacity. Recent work suggests that genetics heavily influence cardiomyocyte ploidy. However, the developmental course by which cardiomyocytes reach their final ploidy state has only been investigated in select backgrounds. Here, we assessed cardiomyocyte number, cell cycle activity, and ploidy dynamics across two divergent mouse strains: C57BL/6J and A/J. Both strains are born and reach adulthood with comparable numbers of cardiomyocytes; however, the end composition of ploidy classes and developmental progression to reach the final state differ substantially. We expand on previous findings that identified Tnni3k as a mediator of cardiomyocyte ploidy and uncover a role for Runx1 in ploidy dynamics and cardiomyocyte cell division, in both developmental and injury contexts. These data provide novel insights into the developmental path to cardiomyocyte polyploidization and challenge the paradigm that hypertrophy is the sole mechanism for growth in the postnatal heart.

The Senolytic Drug Fisetin Attenuates Bone Degeneration in the Zmpste24 -/- Progeria Mouse Model.

Aging leads to several geriatric conditions including osteoporosis (OP) and associated frailty syndrome. Treatments for these conditions are limited and none target fundamental drivers of pathology, and thus identifying strategies to delay progressive loss of tissue homeostasis and functional reserve will significantly improve quality of life in elderly individuals. A fundamental property of aging is the accumulation of senescent cells. Senescence is a cell state defined by loss of proliferative capacity, resistance to apoptosis, and the release of a proinflammatory and anti-regenerative senescence-associated secretory phenotype (SASP). The accumulation of senescent cells and SASP factors is thought to significantly contribute to systemic aging. Senolytics-compounds which selectively target and kill senescent cells-have been characterized to target and inhibit anti-apoptotic pathways that are upregulated during senescence, which can elicit apoptosis in senescent cells and relieve SASP production. Senescent cells have been linked to several age-related pathologies including bone density loss and osteoarthritis in mice. Previous studies in murine models of OP have demonstrated that targeting senescent cells pharmacologically with senolytic drugs can reduce symptomology of the disease. Here, we demonstrate the efficacy of senolytic drugs (dasatinib, quercetin, and fisetin) to improve age-associated degeneration in bone using the Zmpste24-/- (Z24-/-) progeria murine system for Hutchinson-Gilford progeria syndrome (HGPS). We found that the combination of dasatinib plus quercetin could not significantly mitigate trabecular bone loss although fisetin administration could reduce bone density loss in the accelerated aging Z24-/- model. Furthermore, the overt bone density loss observed in the Z24-/- model reported herein highlights the Z24 model as a translational model to recapitulate alterations in bone density associated with advanced age. Consistent with the "geroscience hypothesis," these data demonstrate the utility of targeting a fundamental driver of systemic aging (senescent cell accumulation) to alleviate a common condition with age, bone deterioration.

Attenuation of Graft-Versus-Host-Disease Via Genetic or Pharmacologic Inhibition of the RNA Splicing Factor RBM39

Recent studies have identified pharmacologic modulation of RNA splicing via degradation of the splicing factor RBM39 to mediate anticancer effects and enhance responses to immune checkpoint blockade through generation of novel RNA-splicing derived immunogenic peptides in tumors. However rigorous evaluation of the role of RBM39 in immune cells has not been performed. Here we identify that RBM39 modulates exon recognition in T cells to regulate their function. Although essential for the survival of hematopoietic precursors and tumors, RBM39 is dispensable for peripheral T cell survival. Functional evaluation of the T cell alloantigen response in allogeneic hematopoietic stem transplantation (allo-HSCT) revealed that Rbm39 loss attenuated graft-versus-host-disease (GVHD) and improved overall survival while partially sparing graft-versus-tumor (GVT) effects. To rigorously evaluate the role of RBM39 in immune cells, we generated an Rbm39 conditional knockout mouse by floxing Rbm39 exon 7, which encodes its first RNA recognition motif. Pan-hematopoietic deletion of Rbm39 resulted in rapid complete failure of hematopoiesis and impaired hematopoietic stem cell (HSC) self-renewal. We next analyzed the phenotype of animals with T cell specific deletion of RBM39 and compared this with the effects of pharmacologic RBM39 degradation on T cells. Strikingly, in contrast to the profound effects of genetic RBM39 loss in HSCs and the inability of cancer cells to tolerate RBM39 deletion, RBM39 deletion under CD4-cre (deletion early in thymopoiesis) or distal Lck-cre (late in thymopoiesis) yielded modestly fewer viable peripheral T cells, which skewed towards a CD44+ effector/memory phenotype. This observation was also recapitulated with extended treatment of mice with the RBM39 degrader E7820 (in phase II trials currently; clinicaltrials.gov NCT05024994). Deep RNA-seq analysis of splenic CD3+ T cells from distal Lck-cre Rbm39fl/fl mice revealed widespread decreased efficiency of RNA splicing in Rbm39 null T cells compared to controls, indicative of altered RNA splicing in T cells with Rbm39 deletion. Rbm39 KO T cells experienced global increases in exon skipping at genomic regions where upstream introns had reduced GC content. These data identify that RBM39 is required in a cell and tissue-specific manner for cell survival and further highlights the selectivity of therapeutic approaches targeting RBM39. We next assessed the functional consequences of pharmacologic RBM39 degradation or genetic RBM39 deletion in T cells. Consistent with observations in genetic mouse models, T cells could tolerate extensive pharmacologic RBM39 degradation without loss of proliferative capacity in response to PMA/Ionomycin or anti-CD3+CD28 stimulation. Similarly, genetic Rbm39 null T cells had an intact proliferative response to PMA/Ionomycin. Furthermore, when we evaluated the effects of RBM39 loss in the MHC-mismatched C57BL/6 => Balb/c allo-HSCT system, Rbm39 deletion in donor T cells under either CD4- or distal Lck-cre profoundly reduced GVHD severity and improved overall survival in recipients (Fig. A). These genetic findings were recapitulated via pharmacologic degradation of RBM39 in the same HSCT system (Fig. B). Similar experiments performed in the setting of Balb/c hosts engrafted with A20 lymphoma cells revealed partial preservation of GVT activity using Rbm39 KO T cells. Single cell RNA sequencing analyses of CD4-cre Rbm39fl/fl primary T cells revealed upregulation of the integrin beta1 subunit in both CD4 and CD8 T cells. This suggests alteration of T cell trafficking away from GVHD target organs as one mechanism by which RBM39 loss may alter T cell alloreactivity in allo-HSCT. Our results identify cell-type specific roles for RBM39 in hematopoiesis and immune cells and suggest a therapeutic approach for GVHD via direct modulation of RNA splicing. Of note, as the RBM39 degrader E7820 is currently in phase II clinical trials for relapsed myeloid neoplasms, our previous studies and this data identify a treatment for myeloid neoplasms in the post-transplant setting, where RBM39 degraders mediate both direct and immune-mediated anti-tumor effects while also attenuating GVHD via modulation of RNA splicing in T cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 13491: Spermidine Improves Angiogenic Capacity in Senescent Endothelial Cells and Enhances Ischemia Induced Neovascularization in Aged Mice

Aging is closely associated with high morbidity and mortality of various diseasesincluding cardiovascular disease. Cellular senescence has been cruciallyinvolved in the mechanisms underlying age-related diseases, and vascularendothelial cells <ECs> become senescent during aging. Spermidine, a naturallyoccurring polyamine, is an emerging organic compound that shows various anti-aging effects. Serum spermidine concentration decreases during aging inhuman, and dietary spermidine has been reported to extend healthy lifespanand delay the age-related decline in cognitive and physical performance inmany species such as yeast, worms, flies, and mice. Here, we explored aneffect of spermidine on age-associated decline in angiogenesis. Replicativesenescent ECs were prepared by culturing human ECs for an extended perioduntil passage 18-20, and cellular senescence was confirmed by loss ofproliferation capacity, increased CDK inhibitors expression, and senescence-associated beta-Gal activity. Intracellular polyamine contents were significantlyreduced in replicative senescent ECs, which was recovered by spermidinesupplementation. Spermidine supplementation improved the declinedangiogenic capacities including tube-formation on Matrigel in senescent ECs,while senescence features such as reduced proliferation and senescence-associated beta-Gal activity were not affected. Mechanistically, spermidinesupplementation accelerated the static autophagy flux, and improvedmitochondrial quality, e.g. reduced mitochondrial ROS and preservedmitochondrial membrane potential, in senescent ECs. Ischemia-inducedneovascularization was assessed using the hind-limb ischemia model in youngand aged mice. Blood flow recovery and neovascularization in ischemic musclewere considerably impaired in aged mice comparing to those in young mice. Ofnote, dietary spermidine significantly enhanced ischemia-induced angiogenesis,and improved the blood flow recovery in the ischemic limb, especially in agedmice. Our results reveal new mechanistic insights in anti-aging property ofspermidine, and suggest its therapeutic potential against ischemiccardiovascular disease, particularly in elderly population.

Novel immortalization approach defers senescence of cultured canine adipose-derived mesenchymal stromal cells.

In the last decades, the scientific community spared no effort to elucidate the therapeutic potential of mesenchymal stromal cells (MSCs). Unfortunately, in vitro cellular senescence occurring along with a loss of proliferative capacity is a major drawback in view of future therapeutic applications of these cells in the field of regenerative medicine. Even though insight into the mechanisms of replicative senescence in human medicine has evolved dramatically, knowledge about replicative senescence of canine MSCs is still scarce. Thus, we developed a high-content analysis workflow to simultaneously investigate three important characteristics of senescence in canine adipose-derived MSCs (cAD-MSCs): morphological changes, activation of the cell cycle arrest machinery, and increased activity of the senescence-associated β-galactosidase. We took advantage of this tool to demonstrate that passaging of cAD-MSCs results in the appearance of a senescence phenotype and proliferation arrest. This was partially prevented upon immortalization of these cells using a newly designed PiggyBac™ Transposon System, which allows for the expression of the human polycomb ring finger proto-oncogene BMI1 and the human telomerase reverse transcriptase under the same promotor. Our results indicate that cAD-MSCs immortalized with this new vector maintain their proliferation capacity and differentiation potential for a longer time than untreated cAD-MSCs. This study not only offers a workflow to investigate replicative senescence in eukaryotic cells with a high-content analysis approach but also paves the way for a rapid and effective generation of immortalized MSC lines. This promotes a better understanding of these cells in view of future applications in regenerative medicine.

Abstract 9603: Polycomb Group Protein Cbx7 Represses Cardiomyocyte Proliferation via Modulation of the Tardbp/Rbm38 Axis

Introduction: Cardiomyocyte (CM) proliferation notably decreases during the perinatal period. Regulatory mechanisms for this loss of proliferative capacity are poorly understood. CBX7, a polycomb group (PcG) protein, regulates the cell cycle but its role in CM proliferation is unknown. Methods: We profiled CBX7 expression in the mouse hearts via qRT-PCR, western blotting, and immunohistochemistry. We overexpressed CBX7 in neonatal mouse CMs via adenoviral transduction. We knocked down CBX7 by using constitutive and inducible conditional knockout mice (Myh6-cre;Cbx7 fl/+ and Myh6-MCM;Cbx7 fl/fl , respectively). We measured CM proliferation by immunostaining of proliferation markers such as Ki67, phospho-histone 3, and cyclin B1. We examined the mechanism of CBX7-mediated repression of CM proliferation via co-immunoprecipitation, mass spectrometry, and other molecular techniques. Results: The mRNA expression of Cbx7 was increased at the perinatal stage and sustained in the postnatal heart (fold increase of adult vs. prenatal: 32.1 ± 3.3, P < 0.01). Overexpression of CBX7 in neonatal mouse CMs reduced CM proliferation (% of Ki67 + CMs: Ad-Mock, 22.3 ± 1.7 vs. Ad-CBX7, 7.7 ± 0.7, P < 0.001). The haplodeficiency of CBX7 (Tnnt2-Cre;Cbx7 fl/+ ) enhanced proliferation of neonatal CMs in vivo (% of Ki67 + CMs: wild-type, 7.9 ± 0.9% vs. mutants, 24.7 ± 1.2%, P < 0.01), leading to increased myocardial wall thickness, cardiomegaly and neonatal lethality. Genetic deletion of CBX7 in CMs (Myh6-MCM;Cbx7 fl/fl ) at P1 resulted in increased CM proliferation (% of Ki67 + CMs: vehicle, 9.5 ± 1.4% vs. tamoxifen, 18.3 ± 1.8%, P < 0.01), leading to cardiomegaly at 3 months. Mechanistically, CBX7 interacted with TAR DNA-binding protein 43 (TARDBP) and positively regulated its downstream target, RNA Binding Motif Protein 38 (RBM38). Rbm38 was perinatally upregulated in the mouse hearts and overexpression of RBM38 reduced proliferation of neonatal CMs. Conclusions: CBX7 expression is perinatally increased in the mouse hearts and inhibits proliferation of CMs by controlling TARDBP/Rbm38 pathway. This is the first study to demonstrate the role of CBX7 in regulation of CM proliferation and CBX7 could be an important target for cardiac regeneration.

Adult neural stem cells have latent inflammatory potential that is kept suppressed by Tcf4 to facilitate adult neurogenesis.

Inflammation is known to adversely affect adult neurogenesis, wherein the source of inflammation is largely thought to be extraneous to the neurogenic niche. Here, we demonstrate that the adult hippocampal neural progenitors harbor an inflammatory potential that is proactively suppressed by transcription factor 4 (Tcf4). Deletion of Tcf4 in hippocampal nestin-expressing progenitors causes loss of proliferative capacity and acquisition of myeloid inflammatory properties. This transformation abolishes their differentiation potential and causes production of detrimental factors that adversely affect niche cells, causing inflammation in the dentate gyrus. Thus, on one hand, Tcf4 deletion causes abrogation of proliferative progenitors leading to reduction of adult neurogenesis, while on the other, their accompanying inflammatory transformation inflicts inflammation in the niche. Taken together, we provide the first evidence for a latent inflammatory potential of adult hippocampal neural progenitors and identify Tcf4 as a critical regulator that facilitates adult neurogenesis via proactive suppression of this detrimental potential.

Premature senescence of placental decidua cells as a possible cause of miscarriage produced by mycophenolic acid

BackgroundSuccessful pregnancy is supported by a healthy maternal–fetal interface (i.e., the decidual tissues) which holds the conceptus and safeguards it against stressors from the beginning of pregnancy. Any disturbance of this interface can presumably lead to the loss of pregnancy. The use of the immunosuppressive drug mycophenolic acid (MPA) should be discontinued in pregnancy given its abortive and embryotoxic effects. Direct teratogenic effects have been observed in mammalian embryos cultured in MPA, but the underlying mechanisms of abortion by MPA are less understood.MethodsDecidual stromal cells isolated from human placentas are cultured in the presence of clinically relevant doses of MPA. Data regarding the effects of MPA on the proliferation and viability of decidua cultures are first analysed and then, molecular pathways contributing to these effects are unravelled.ResultsMPA treatment of decidual stromal cells results in loss of proliferation capacity and a decrease in the viability of decidua cultures. The molecular pathways involved in the effects of MPA on decidual stromal cells are a reduction in pre-rRNA synthesis and subsequent disruption of the nucleolus. The nucleolar stress stabilizes p53, which in turn, leads to a p21–mediated cell cycle arrest in late S and G2 phases, preventing the progression of the decidua cells into the mitosis. Furthermore, MPA does not induce apoptosis but activate mechanisms of autophagy and senescence in decidual stromal cells.ConclusionThe irreversible growth arrest of decidua cells, whose role in the maintenance of the pregnancy microenvironment is known, may be one cause of miscarriage in MPA treated pregnant women.

Abstract 5831: Clearance of therapy-induced senescent cancer cells by the senolytic ABT-263 in a model of non-small cell lung cancer

Abstract While most chemotherapies will initially lead to tumor reduction in patients, residual tumor cells that enter a state of dormancy can re-emerge as aggressive, recurrent disease after treatment is terminated. One mechanism by which cancer cells may escape chemotherapy-induced death is senescence. Senescence is a specialized form of cell cycle arrest that is primarily characterized as a durable, prolonged loss of proliferative capacity. While senescence was traditionally considered to be irreversible, evidence has accumulated in recent years supporting the premise that senescence is instead a durable form of growth arrest from which a subpopulation of cells can ultimately escape. Moreover, the accumulation of senescent cells post cancer therapy has been linked with a plethora of unfavorable outcomes. A sequential approach in which chemotherapy is followed by a senolytic, an emerging class of drugs that selectively kill senescent cells, may therefore delay or circumvent recurrent disease by eliminating residual senescent cells. To this end, we tested whether etoposide- or radiation-treated non-small cell lung cancer (NSCL) cells become susceptible to the BH-3 mimetic ABT-263– an established senolytic– following senescence induction. Treatment with etoposide or radiation induced a robust senescent state, as evidenced by the development of multiple senescent phenotypes. Senescent tumor cells demonstrated increased sensitivity to ABT-263 compared to non-senescent cells, undergoing rapid apoptosis subsequent to ABT-263 exposure. We further determined that ABT-263's mechanism of action appears to depend upon the disruption of the BCL-XL/Bax interaction. ABT-263's capacity to eliminate senescent cells is conserved in vivo, as tumor-bearing animals treated with etoposide followed by ABT-263 undergo rapid tumor reduction followed by tumor maintenance, while etoposide-only treated animals develop progressive disease subsequent to treatment completion. These data support the use of senolytics as complementary therapy to eliminate residual tumor cells following chemotherapy or radiation, and thereby potentially delay or reduce recurrent disease. Citation Format: Valerie Carpenter, Tareq Saleh, David Gewirtz. Clearance of therapy-induced senescent cancer cells by the senolytic ABT-263 in a model of non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5831.

Induction of different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells treated with various doses of 177Lu-Nimotuzumab

Introduction Radioimmunotherapy (RIT) is a major anti-cancer therapy in cancer management multimodalities. 177Lu-Nimotuzumab has been in the use for radioimmunotherapy of EGFR expressing tumors. This study focuses on understanding the differential cellular and molecular mechanisms of anti-tumor effects of 177Lu-Nimotuzumab on low EGFR expressing A549 and high EGFR expressing A431 tumor cells. Materials and methods Nimotuzumab labeled with 177Lu was characterized by SE-HPLC. Specificity of 177Lu-Nimotuzumab to EGFR expressed on A549 and A431 cells was confirmed by competitive assay using increasing amounts of unlabeled Nimotuzumab. Cellular responses of A549 (low EGFR) and A431 (high EGFR) in response to different doses of 177Lu-Nimotuzumab were determined by Viable count assay for cellular viability, cell-cycle analysis by DNA staining, apoptotic assay for cell death, and CFSE dilution assay for cellular proliferation capacity. The number of DNA DSBs formed was determined using γ-H2AX assay with PI staining. Transcription of genes involved in DNA damage response and repair (DRR) pathways was monitored by RT-qPCR. Results 177Lu-Nimotuzumab characterized by SE-HPLC exhibited a radiochemical purity of 99.1 ± 0.6%. Cell binding competition studies with 177Lu-Nimotuzumab showed specific binding of 34.3 ± 1.7% with A431 cells and 18.4 ± 1.9% with A549 cells which decreased when co-incubated with unlabeled Nimotuzumab. Cytotoxicity and DNA damage (DNA DSBs) increased with an increase in the dose of 177Lu-Nimotuzumab. A549 displayed G2/M arrest while A431 showed G1 arrest. Apoptotic death was determined to be one of the modes of death of arrested A549 and A431 cells which increases with the increase in the dose of 177Lu-Nimotuzumab. Loss of proliferation capacity was higher in A431 showed by CFSE staining at different doses of 177Lu-Nimotuzumab. Transcription profile of most DRR genes in A431 and A549 showed a decrease in the transcription at 4 h followed by recovery at 16 h post-treatment. The degree of transcription of most DRR genes was similar, irrespective of 177Lu-Nimotuzumab dose. Conclusion 177Lu-Nimotuzumab induces different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells. This study would enable the development of integrative novel treatment strategies for radioimmunotherapy in low and high EGFR expressing tumors by 177Lu-Nimotuzumab with therapeutic gains.

The senolytic drug fisetin mitigates age‐related bone density loss in the progeroid mouse model Zmpste24−/−

Senescence is a cell state defined by loss of proliferative capacity, increased metabolic activity, and importantly, resistance to apoptosis. Senescent cells can release pro‐inflammatory cytokines/chemokines, proteases, and other factors, otherwise known as the senescence‐associated secretory phenotype (SASP). The SASP can adversely affect not only neighboring cells, but also likely contributes to driving systemic aging. Senolytic compounds such as dasatinib (D), quercetin (Q), and fisetin (FIS) were recently found to target anti‐apoptotic pathways known to be upregulated in senescent cells. Several reports have found significant improvement in healthspan indices including decreased frailty, neurologic dysfunction, and bone loss in vivo following intermittent treatment with senolytic agents. Our lab routinely uses the accelerated aging model Zmpste24−/− (Z24−/−) mice to investigate age‐related pathologies as they have a maximum lifespan of ~6 months. For the first time here, we examined the effects of another significantly safer senolytic compound, the flavonoid FIS, on spontaneous bone density loss associated with age using the Z24−/− model.Z24−/− mice begin to exhibit significant musculoskeletal decline associated with increased senescent cell burden as early as 2 months including weight loss and reduced cartilage proteoglycan and bone levels in skeletal preparations (Fig. 1). Importantly, we previously found that senolytic drug treatment restored cartilage in Z24−/− mice in a dose dependent manner. These data demonstrated for the first time the therapeutic potential for senotherapuetic drugs for the treatment of age‐associated knee OA. Fisetin, a newly characterized senolytic drug was recently demonstrated to significantly extend lifespan and healthspan, including musculoskeletal function, in progeroid mice. We thus next evaluated if FIS, a less toxic alternative to dasatinib that targets several senescence associated pathways, might better prevent bone loss in rapidly aging Z24−/− mice. Indeed, we found that weekly dosing of FIS for weeks starting at 3 months of age could significantly attenuate bone density loss in Z24−/− mice as evidenced by micro‐CT analysis (Fig. 2). Future studies will examine if more chronic dosing of FIS at younger ages.Aging is associated with sever musculoskeletal decline including OA and osteoporosis for which there are few treatment options none of which that target shared fundamental mechanistic drivers. We previously found that the senolytic drug cocktail DQ could prevent articular cartilage degeneration. The main objective of these studies was to investigate the efficacy of senolytic drugs, principally FIS, to attenuate age‐associated loss of bone density in progeroid Z24−/− mice. In agreement to previous reports, we found senolytic drug treatment could mitigate bone density loss in aged/progeroid mice. However, we report this for the first time using FIS, a much safer alternative to chemotherapeutic agents with similar efficacy. FIS may thus be a more clinically viable option for senotherapeutic treatment.Support or Funding InformationNIA (5P01AG043376‐05).Musculoskeletal pathology in Z24−/− mice is improved with DQ treatmentFigure 1Effects of senolytic drugs on trabecular bone in Z24−/− miceFigure 2

Interventional Strategies to Delay Aging Related Diseases & Conditions of the Musculoskeletal System

Aging leads to several geriatric syndromes including frailty, a condition characterized by loss of functional reserve and tissue regeneration repair capacity. Frail individuals exhibit significant mobility and psychological deficits resulting in significant healthcare costs. Thus, identifying strategies to delay aging, or prevent the progressive loss of tissue homeostasis and functional reserve associated with frailty, will dramatically restore function and independence in millions of elderly patients and significantly improve quality of life. We have demonstrated that bone marrow‐derived mesenchymal stem cells (MSCs) and muscle‐derived stem/progenitor cells (MDSPCs) become dysfunctional with age. A fundamental property of aging is the accumulation of senescent cells. Senescence is a cell state defined by loss of proliferative capacity, increased metabolic activity, and importantly, resistance to apoptosis. Senescent cells can release pro‐inflammatory cytokines/chemokines, proteases, and other factors known as the senescence‐associated secretory phenotype (SASP). The SASP can adversely affect not only neighboring cells, but also likely contributes to driving systemic aging. Compounds that selectively target and kill senescent cells were recently identified and characterized. These senolytic drugs target and inhibit anti‐apoptotic pathways that are upregulated in senescent cells thereby inducing apoptotic cell death and abrogating systemic SASP factors. Importantly, senolytic drugs have a favorable safety profile being either FDA approved (dasatinib) or over the counter flavonoid supplements (quercetin, fisetin), positive findings may be more readily translatable to Phase I–II clinical trials. Further, it has been reported that exposure to factors present in the serum of young mice restores the regenerative capacity of aged progenitor cells, through parabiotic pairing. We believe that pregnancy represents a unique biological model of a naturally‐shared circulatory system between young and old organisms. Our preliminary data have demonstrated improved musculoskeletal tissue healing after injury in pregnant mice and improved differentiation capacity of aged muscle progenitor cells (MPCs) after stimulation with serum from pregnant mice. We hypothesize that circulating factors from fetal microchimeric cells (FMCs) during pregnancy have beneficial effects on maternal wound healing, such as reduction in fibrosis for optimal tissue regeneration. In summary, we believe these novel interventional strategies could be used to restore the endogenous stem cell populations in age‐related disorderst to promote healthy aging & to delay age‐related disorders.

SLAMF6 as a Regulator of Exhausted CD8+ T Cells in Cancer.

The tumor microenvironment in leukemia and solid tumors induces a shift of activated CD8+ cytotoxic T cells to an exhausted state, characterized by loss of proliferative capacity and impaired immunologic synapse formation. Efficient strategies and targets need to be identified to overcome T-cell exhaustion and further improve overall responses in the clinic. Here, we took advantage of the Eμ-TCL1 chronic lymphocytic leukemia (CLL) and B16 melanoma mouse models to assess the role of the homophilic cell-surface receptor SLAMF6 as an immune-checkpoint regulator. The transfer of SLAMF6+ Eμ-TCL1 cells into SLAMF6-/- recipients, in contrast to wild-type (WT) recipients, significantly induced expansion of a PD-1+ subpopulation among CD3+CD44+CD8+ T cells, which had impaired cytotoxic functions. Conversely, administering anti-SLAMF6 significantly reduced the leukemic burden in Eμ-TCL1 recipient WT mice concomitantly with a loss of PD-1+CD3+CD44+CD8+ T cells with significantly increased effector functions. Anti-SLAMF6 significantly reduced leukemic burden in the peritoneal cavity, a niche where antibody-dependent cellular cytotoxicity (ADCC) is impaired, possibly through activation of CD8+ T cells. Targeting of SLAMF6 affected tumor growth not only in B cell-related leukemia and lymphomas but also in nonhematopoietic tumors such as B16 melanoma, where SLAMF6 is not expressed. In vitro exhausted CD8+ T cells showed increased degranulation when anti-human SLAMF6 was added in culture. Taken together, anti-SLAMF6 both effectively corrected CD8+ T-cell dysfunction and had a direct effect on tumor progression. The outcomes of our studies suggest that targeting SLAMF6 is a potential therapeutic strategy.