Quiescent Cells Research Articles

Abstract 2119: Transcriptomic Profiling Reveals Therapeutic Potential Of Docosahexaenoic Acid For SARS Coronaviruses Infection Management

Prior work from our laboratory not only suggested that docosahexaenoic acid (DHA) may upregulate immune response pathways in human monocytes, it also revealed that DHA reduces angiotensin converting enzyme 2 (ACE2), the cellular receptor for SARS coronaviruses (SARS-CoV), in various rat tissues and human cultured cells. Also, DHA was found to inhibit cellular entry of SARS-CoV-2 pseudovirus. Thus, it was hypothesized that DHA has the potential to help manage SARS-CoV infection. RNA-seq was performed on DHA-treated (20 or 125 μM for 8 h) and control human EA.hy926 cells in both the growing and quiescent states. The data were processed by the RSEM-STAR-DESeq2 pipeline, and then subjected to gene set enrichment analysis (GSEA) with clusterProfiler. GSEA revealed that only in quiescent cells, 20 μM DHA downregulated pathways related to SARS-CoV-1/2-host interactions, specifically the processes by which the virus disrupts host protein translation and global mRNA splicing to suppress host defenses. The Reactome term “potential therapeutics for SARS” was positively enriched by both 20 and 125 μM DHA in quiescent cells only, including genes related to nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, interleukin-6 pathway, heat shock proteins, and TBK1 . However, in growing cells, terms related to SARS-CoV-1/2-host interactions were upregulated by DHA. Beyond derailing SARS-CoV via translation machinery and ACE2, DHA was also previously found to concomitantly reduce ACE1 protein levels, thus preserving the ACE1/ACE2 balance. This is significant because the balance of ACE1/ACE2 is important for maintaining renin-angiotensin system homeostasis, a factor critical in the pathogenesis of long COVID. Overall, our findings advance a novel perspective on the therapeutic potential of DHA for managing SARS-CoV infection, via its ability to hinder virus-host interactions. Moreover, the beneficial effects of DHA only occurred in quiescent (healthy) endothelial cells but not growing (dysfunctional) endothelial cells, implying that COVID-19 patients without CVD may be more responsive to DHA treatment compared to patients with underlying CVD. Further in vitro , in vivo , and even clinical studies are required to validate these effects of DHA on SARS-CoV.

How do 2'-O-methylations within Human Immunodeficiency Virus type 1 (HIV-1) genome regulate its replication?

The genomic RNA of HIV-1 is modified by epitranscriptomic modifications, including 2'-O-methylations, which are found on 17 internal positions. These methylations are added by the cellular methyltransferase FTSJ3, and have pro-viral effects, since they shield the viral genome from the detection by the innate immune sensor MDA5. In turn, the production of interferons by infected cells is reduced, limiting the expression of interferon-stimulated genes (ISGs) with antiviral activities. Moreover, 2'-O-methylations protect the HIV-1 genome from its degradation by ISG20, an interferon-induced exonuclease. Conversely, these methylations also exhibit antiviral effects, as they impede reverse-transcription in vitro or in quiescent cells, which are known to contain low nucleotide concentrations. Altogether, these observations suggest a balance between the proviral effect of 2'-O-methylations, related to the protection of the viral genome from detection by MDA5 and degradation by ISG20, and the antiviral effect, associated with the negative impact of 2'-O-methylations on the viral replication. These findings pave the way for further optimization of therapeutic RNA, by selective methylation of specific nucleotides.

Hydrogen Peroxide Signaling in the Maintenance of Plant Root Apical Meristem Activity.

Hydrogen peroxide (H2O2) is a prevalent reactive oxygen species (ROS) found in cells and takes a central role in plant development and stress adaptation. The root apical meristem (RAM) has evolved strong plasticity to adapt to complex and changing environmental conditions. Recent advances have made great progress in explaining the mechanism of key factors, such as auxin, WUSCHEL-RELATED HOMEOBOX 5 (WOX5), PLETHORA (PLT), SHORTROOT (SHR), and SCARECROW (SCR), in the regulation of RAM activity maintenance. H2O2 functions as an emerging signaling molecule to control the quiescent center (QC) specification and stem cell niche (SCN) activity. Auxin is a key signal for the regulation of RAM maintenance, which largely depends on the formation of auxin regional gradients. H2O2 regulates the auxin gradients by the modulation of intercellular transport. H2O2 also modulates the expression of WOX5, PLTs, SHR, and SCR to maintain RAM activity. The present review is dedicated to summarizing the key factors in the regulation of RAM activity and discussing the signaling transduction of H2O2 in the maintenance of RAM activity. H2O2 is a significant signal for plant development and environmental adaptation.

Assessment of Redox Patterns at the Transcriptional and Systemic Levels in Newly Diagnosed Acute Leukemia

Background: Acute leukemia is the result of clonal transformation and proliferation of a hematopoietic progenitor giving rise to poorly differentiated neoplastic cells. Reactive oxygen species play a role in maintaining the quiescence, self-renewal, and long-term survival of hematopoietic stem cells, but it is unclear how they would affect disease onset and progression. The aim is to evaluate, at the transcriptional and systemic level, the oxidative-inflammatory status in newly diagnosis acute leukemia patients. Methods: Seventy acute leukemia patients [26 acute lymphoblastic leukemia (ALL), 13 Acute Promyelocytic Leukemia (APL), and 31 Acute Myeloid Leukemia (AML)] and forty-one healthy controls were analyzed. Malondialdehyde and catalase activity were evaluated. Gene expression of NRF2, SOD, PRDX2, CAT, IL-6, and TNF-α was analyzed by real-time PCR. Results: Malondialdehyde concentration was similar in all groups studied. Catalase activity was significantly higher in AML and APL patients compared to controls, while ALL showed similar activity to the healthy group. NRF2, CAT, and PRDX2 expression levels were similar between groups, SOD expression was downregulated in all acute leukemia patients. TNF-α expression was lower in AML groups than in healthy individuals, and IL-6 mRNA expression was downregulated in ALL and APL. Conclusion: This is the first report that correlates transcriptional and systemic parameters associated with the oxidative inflammatory status in newly diagnosed acute leukemia. Some of the parameters evaluated could be used as biomarkers in the selection of an effective therapeutic strategy and will open new directions for the follow-up and evolution of this disease.

Redox regulation of meristem quiescence: outside/in.

Quiescence is an essential property of meristematic cells, which restrains the cell cycle while retaining the capacity to divide. This crucial process not only facilitates life-long tissue homeostasis and regenerative capacity but also provides protection against adverse environmental conditions, enabling cells to conserve the proliferative potency while minimizing DNA damage. As a survival attribute, quiescence is inherently regulated by the products of aerobic life, in particular reactive oxygen species (ROS) and the redox (reduction/oxidation) mechanisms that plant have evolved to channel these into pervasive signals. Adaptive responses allow quiescent cells to compensate for reduced oxygen tension (hypoxia) in a reversible manner, while the regulated production of the superoxide anion (O2·-) facilitates cell division and the maintenance of stem cells. Here we discuss the role of ROS and redox reactions in the control of the quiescent state in plant meristems, and how this process is integrated with cellular energy and hormone biochemistry. We consider the pathways that sense and transmit redox signals with a focus on the central significance of redox regulation in the mitochondria and nucleus, which is a major regulator of quiescence in meristems. We discuss recent studies that suggest that ROS are a critical component of the feedback loops that control stem cell identity and fate, and suggest that the ROS/hypoxia interface is an important 'outside/in' positional cue for plant cells, particularly in meristems.

The potential role of CMC1 as an immunometabolic checkpoint in T cell immunity

ABSTRACT T cell immunity is critical for human defensive immune response. Exploring the key molecules during the process provides new targets for T cell-based immunotherapies. CMC1 is a mitochondrial electron transport chain (ETC) complex IV chaperon protein. By establishing in-vitro cell culture system and Cmc1 gene knock out mice, we evaluated the role of CMC1 in T cell activation and differentiation. The B16-OVA tumor model was used to test the possibility of targeting CMC1 for improving T cell anti-tumor immunity. We identified CMC1 as a positive regulator in CD8+T cells activation and terminal differentiation. Meanwhile, we found that CMC1 increasingly expressed in exhausted T (Tex) cells. Genetic lost of Cmc1 inhibits the development of CD8+T cell exhaustion in mice. Instead, deletion of Cmc1 in T cells prompts cells to differentiate into metabolically and functionally quiescent cells with increased memory-like features and tolerance to cell death upon repetitive or prolonged T cell receptor (TCR) stimulation. Further, the in-vitro mechanistic study revealed that environmental lactate enhances CMC1 expression by inducing USP7, mediated stabilization and de-ubiquitination of CMC1 protein, in which a mechanism we propose here that the lactate-enriched tumor microenvironment (TME) drives CD8+TILs dysfunction through CMC1 regulatory effects on T cells. Taken together, our study unraveled the novel role of CMC1 as a T cell regulator and its possibility to be utilized for anti-tumor immunotherapy.

Molecular heterogeneity of quiescent melanocyte stem cells revealed by single-cell RNA-sequencing.

Melanocyte stem cells (McSCs) of the hair follicle are a rare cell population within the skin and are notably underrepresented in whole-skin, single-cell RNA sequencing (scRNA-seq) datasets. Using a cell enrichment strategy to isolate KIT+/CD45- cells from the telogen skin of adult female C57BL/6J mice, we evaluated the transcriptional landscape of quiescent McSCs (qMcSCs) at high resolution. Through this evaluation, we confirmed existing molecular signatures for qMcCS subpopulations (e.g., Kit+, Cd34+/-, Plp1+, Cd274+/-, Thy1+, Cdh3+/-) and identified novel qMcSC subpopulations, including two that differentially regulate their immune privilege status. Within qMcSC subpopulations, we also predicted melanocyte differentiation potential, neural crest potential, and quiescence depth. Taken together, the results demonstrate that the qMcSC population is heterogeneous and future studies focused on investigating changes in qMcSCs should consider changes in subpopulation composition.

Mirk/Dyrk1B Kinase Inhibitors in Targeted Cancer Therapy.

During the last years, there has been an increased effort in the discovery of selective and potent kinase inhibitors for targeted cancer therapy. Kinase inhibitors exhibit less toxicity compared to conventional chemotherapy, and several have entered the market. Mirk/Dyrk1B kinase is a promising pharmacological target in cancer since it is overexpressed in many tumors, and its overexpression is correlated with patients' poor prognosis. Mirk/Dyrk1B acts as a negative cell cycle regulator, maintaining the survival of quiescent cancer cells and conferring their resistance to chemotherapies. Many studies have demonstrated the valuable therapeutic effect of Mirk/Dyrk1B inhibitors in cancer cell lines, mouse xenografts, and patient-derived 3D-organoids, providing a perspective for entering clinical trials. Since the majority of Mirk/Dyrk1B inhibitors target the highly conserved ATP-binding site, they exhibit off-target effects with other kinases, especially with the highly similar Dyrk1A. In this review, apart from summarizing the data establishing Dyrk1B as a therapeutic target in cancer, we highlight the most potent Mirk/Dyrk1B inhibitors recently reported. We also discuss the limitations and perspectives for the structure-based design of Mirk/Dyrk1B potent and highly selective inhibitors based on the accumulated structural data of Dyrk1A and the recent crystal structure of Dyrk1B with AZ191 inhibitor.

Endothelial Cell Flow-Mediated Quiescence Is Temporally Regulated and Utilizes the Cell Cycle Inhibitor p27.

Endothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. Primary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time-to-cell cycle reentry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA-seq (single-cell RNA sequencing) analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. Arterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous flow-exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. Endothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence misregulation that leads to vascular dysfunction and disease.

Role of Formoterol on Myogenic Regulatory Factor to Ameliorate Statin-induced Myopathy

Objective: To investigate the effects of β2-adrenergic agonist formoterol on expression of myogenic regulatory factor myogenin in statin-induced myopathies in rats
 Methods: Adult male Sprague-Dawley rats were randomized into control (A), statin-only (B), and statin + formoterol groups (C) (n=30 per group). The control group A received no treatment. B group received simvastatin at 60 mg/kg/day by oral gavage for 12 weeks to induce myotoxicity. C group received simvastatin at 60 mg/kg/day plus formoterol at 3 μg/kg/day by oral gavage for 12 weeks. After 12 weeks, extensor digitorum longus muscles were dissected and 5 μm transverse sections were immunostained for myogenin expression. One cross section was selected from each of the specimen for study. Myogenin-positive nuclei were quantified in 8 random 40x magnification fields per muscle section by a blinded investigator. Data was analyzed using IBM SPSS.
 Results: Myogenin-positive nuclei were minimal in the control (A) group and statin-only (B) groups. In contrast, the statin + formoterol (C) group exhibited a significant increase in myogenin-positive nuclei compared to both control and statin-only groups (p<0.001), indicating enhanced muscle regeneration.
 Conclusion: Formoterol treatment augmented skeletal muscle repair pathways in a rat model of statin-induced myopathy, potentially via activation of quiescent muscle satellite cells and upregulation of myogenic regulatory factor myogenin in group C.

Targeting Unique Features of Quiescent Cancer Stem Cells to Overcome Resistance and Recurrence in Cancer Therapy: A Review

Quiescent cancer stem cells (QCSC) are non-proliferating cells that survive in the G0 phase and have low ki-67 expression and high p27 expression. QCSCs have the ability to evade most chemotherapy, and some subsequent treatments may result in a higher proportion of quiescent cancer stem cells in the tumor. QCSCs are also associated with cancer recurrence rates because they can re-enter the cell cycle to proliferate when tumor environmental conditions are favorable. QCSCs cause high rates of drug resistance and tumor recurrence, therefore it is necessary to understand the properties of QCSCs. QCSCs have a mechanism that regulates the transition between the proliferative phase and the stationary phase in cancer cells, therefore it is necessary to find new treatments to eliminate QCSCs in tumors. In this review, the authors discuss the mechanisms of QCSCs in inducing drug resistance and tumor recurrence as well as therapies to target QCSCs so that the rate of drug resistance and tumor recurrence can be reduced, including in this review: (i) identifying reactive quiescent cancer cells and eliminating them through anticancer reagents. cell cycle dependent; (ii) modulating the transition from the quiescent to the proliferative phase; and (iii) eliminate QCSC by targeting its unique features. Targeting cancer cells that are in proliferating and stationary phase may ultimately be used as a more effective therapeutic strategy for cancer treatment.

Nucleoside supplements as treatments for mitochondrial DNA depletion syndrome.

Introduction: In mitochondrial DNA (mtDNA) depletion syndrome (MDS), patients cannot maintain sufficient mtDNA for their energy needs. MDS presentations range from infantile encephalopathy with hepatopathy (Alpers syndrome) to adult chronic progressive external ophthalmoplegia. Most are caused by nucleotide imbalance or by defects in the mtDNA replisome. There is currently no curative treatment available. Nucleoside therapy is a promising experimental treatment for TK2 deficiency, where patients are supplemented with exogenous deoxypyrimidines. We aimed to explore the benefits of nucleoside supplementation in POLG and TWNK deficient fibroblasts. Methods: We used high-content fluorescence microscopy with software-based image analysis to assay mtDNA content and membrane potential quantitatively, using vital dyes PicoGreen and MitoTracker Red CMXRos respectively. We tested the effect of 15 combinations (A, T, G, C, AT, AC, AG, CT, CG, GT, ATC, ATG, AGC, TGC, ATGC) of deoxynucleoside supplements on mtDNA content of fibroblasts derived from four patients with MDS (POLG1, POLG2, DGUOK, TWNK) in both a replicating (10% dialysed FCS) and quiescent (0.1% dialysed FCS) state. We used qPCR to measure mtDNA content of supplemented and non-supplemented fibroblasts following mtDNA depletion using 20µM ddC and after 14- and 21-day recovery in a quiescent state. Results: Nucleoside treatments at 200µM that significantly increased mtDNA content also significantly reduced the number of cells remaining in culture after 7days of treatment, as well as mitochondrial membrane potential. These toxic effects were abolished by reducing the concentration of nucleosides to 50µM. In POLG1 and TWNK cells the combination of ATGC treatment increased mtDNA content the most after 7days in non-replicating cells. ATGC nucleoside combination significantly increased the rate of mtDNA recovery in quiescent POLG1 cells following mtDNA depletion by ddC. Conclusion: High-content imaging enabled us to link mtDNA copy number with key read-outs linked to patient wellbeing. Elevated G increased mtDNA copy number but severely impaired fibroblast growth, potentially by inhibiting purine synthesis and/or causing replication stress. Combinations of nucleosides ATGC, T, or TC, benefited growth of cells harbouring POLG mutations. These combinations, one of which reflects a commercially available preparation, could be explored further for treatment of POLG patients.

Following the p63/Keratin5 basal cells in the sensory and non-sensory epithelia of the vomeronasal organ.

The vomeronasal organ (VNO) is a part of the accessory olfactory system, which detects pheromones and chemical factors that trigger a spectrum of sexual and social behaviors. The vomeronasal epithelium (VNE) shares several features with the epithelium of the main olfactory epithelium (MOE). However, it is a distinct neuroepithelium populated by chemosensory neurons that differ from the olfactory sensory neurons in cellular structure, receptor expression, and connectivity. The vomeronasal organ of rodents comprises a sensory epithelium (SE) and a thin non-sensory epithelium (NSE) that morphologically resembles the respiratory epithelium. Sox2-positive cells have been previously identified as the stem cell population that gives rise to neuronal progenitors in MOE and VNE. In addition, the MOE also comprises p63 positive horizontal basal cells, a second pool of quiescent stem cells that become active in response to injury. Immunolabeling against the transcription factor p63, Keratin-5 (Krt5), Krt14, NrCAM, and Krt5Cre tracing experiments highlighted the existence of horizontal basal cells distributed along the basal lamina of SE of the VNO. Single cell sequencing and genetic lineage tracing suggest that the vomeronasal horizontal basal cells arise from basal progenitors at the boundary between the SE and NSE proximal to the marginal zones. Moreover, our experiments revealed that the NSE of rodents is, like the respiratory epithelium, a stratified epithelium where the p63/Krt5+ basal progenitor cells self-replicate and give rise to the apical columnar cells facing the lumen of the VNO.

In Vitro Assessment of Cardiac Fibroblast Activation at Physiologic Stiffness.

Cardiac fibroblasts (CF) are an essential cell type in cardiac physiology, playing diverse roles in maintaining structural integrity, extracellular matrix (ECM) synthesis, and tissue repair. Under normal conditions, these cells reside in the interstitium in a quiescent state poised to sense and respond to injury by synthesizing and secreting collagen, vimentin, hyaluronan, and other ECM components. In response to mechanical and chemical stimuli, these "resident" fibroblasts can undergo a transformation through a continuum of activation states into what is commonly known as a "myofibroblast," in a process critical for injury response. Despite progress in understanding the contribution of fibroblasts to cardiac health and disease, much remains unknown about the signaling mediating this activation, in part owing to technical challenges in evaluating CF function and activation status in vitro. Given their role in monitoring the ECM, CFs are acutely sensitive to stiffness and pressure. High basal activation of isolated CFs is common due to the super-physiologic stiffness of traditional cell culture substrates, making assays dependent on quiescent cells challenging. To overcome this problem, cell culture parameters must be tightly controlled, and the use of dishes coated with biocompatible reduced-stiffness substrates, such as 8-kPa polydimethylsiloxane (PDMS), has shown promise in reducing basal activation of fibroblasts. Here, we describe cell culture protocol for maintaining CF quiescence in vitro to enable a dynamic range for the assessment of activation status in response to fibrogenic stimuli using PDMS-coated coverslips. Our protocol provides a cost-effective tool to study fibroblast signaling and activity, allowing researchers to better understand the underlying mechanisms involved in cardiac fibrosis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 8-kPa polydimethylsiloxane (PDMS)/gelatin-coated coverslips for cardiac fibroblast cell culture Basic Protocol 2: Isolation of adult cardiac fibroblasts and plating onto PDMS coverslips Basic Protocol 3: Assessment of cardiac fibroblast activation by α smooth muscle actin (αSMA) immunocytochemistry.

Decoding lifespan secrets: the role of the gonad in Caenorhabditis elegans aging.

The gonad has become a central organ for understanding aging in C. elegans, as removing the proliferating stem cells in the germline results in significant lifespan extension. Similarly, when starvation in late larval stages leads to the quiescence of germline stem cells the adult nematode enters reproductive diapause, associated with an extended lifespan. This review summarizes recent advancements in identifying the mechanisms behind gonad-mediated lifespan extension, including comparisons with other nematodes and the role of lipid signaling and transcriptional changes. Given that the gonad also mediates lifespan regulation in other invertebrates and vertebrates, elucidating the underlying mechanisms may help to gain new insights into the mechanisms and evolution of aging.

Abstract 2782: Characterization of quiescence-associated secretory profile (QuASP) in cancer: A novel defining feature and therapeutic target

Abstract Senescent cells have a unique senescence-associated secretory profile (SASP) and understanding SASP has contributed to numerous advances in knowledge on aging and cancer biology. Quiescence, which is an important yet poorly understood mechanism for chemoresistance, is related but distinct from senescence. Quiescence shares a non-proliferative feature with senescence but is a reversible state in which quiescent cells can reenter the cell cycle. We hypothesized that, analogous to senescent cells, quiescent cells have a distinct quiescence-associated secretory profile (QuASP) and that defining the QuASP will uncover new genes and pathways associated with cancer cell viability and therapeutic resistance. Indeed, suggesting quiescent cells have a unique and biologically important secretome, we found that tumor conditioned medium (TCM) from quiescent ovarian cancer cells (qOvCa) vs. TCM from proliferating cancer cells increased chemoresistance. Similarly, TCM from quiescent cancer cells vs. TCM from proliferating cancer cells suppressed T cell proliferation and INF-gamma/Granzyme B secretion, suggesting a QuASP immunosuppressive effect. RNASeq analysis of qOvCa cells revealed the secreted factor Follistatin (FST) is upregulated in qOvCa and acted as a paracrine pattern to induce chemoresistance. Furthermore, FST was found to be upregulated in peritumoral fluid following chemotherapy and FST levels in patients with OvCa predicted poor prognosis. To identify additional QuASP factors, we used an ER-BIOID labeled system and LC-MS/MS to characterize the secreted proteins from qOvCa induced by serum starvation compared with normal cells in three OvCa cell lines (PT412, PT340, HEY1). Quiescent OvCa cells indeed have a secretome that is highly unique from proliferating cells, with 137/577 upregulated proteins (LFC>1) and 65/577 downregulated proteins (LFC←1 or no detection in quiescence). Several proteins identified as upregulated have been linked to immunosuppression and/or chemoresistance, oncogenesis and cell cycle were selected for validation and further analysis (CLU, VGF, SERPINE1). In conclusion, we find quiescent cells have a unique quiescence associated secretory profile and that the QuASP induces chemoresistance and immunosuppression. As such, this study has important implications for cancer therapy. Citation Format: Qi Jiang, Santiago Panesso-Gómez, Divya Natesan, Matthew L. MacDonald, Ronald J. Buckanovich. Characterization of quiescence-associated secretory profile (QuASP) in cancer: A novel defining feature and therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2782.

Abstract 5906: A new combinatorial therapy that synergistically targets the NuRD complex and proteostasis to eradicate quiescent ovarian cancer cells

Abstract Despite advances in immune and targeted therapies, chemotherapy remains the primary treatment for ovarian cancer (OvCa) patients. Unfortunately, most OvCa patients develop chemotherapy-resistant disease that is inevitably fatal. Quiescence is an important yet poorly understood mechanism underlying chemotherapy resistance. Therefore, we aimed to characterize quiescent ovarian cancer (qOvCa) cells to identify novel therapeutic targets to improve OvCa patient outcomes. Primary patient-derived qOvCa cells and proliferating cells were isolated using a single-cell microfluidics culture device and collected for single-cell RNA-seq. scRNA-seq of qOvCa cells identified (i) downregulation of MBD3 and CHD4, which are components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, and (ii) upregulation of proteasome-associated genes. Knockdown of either MBD3 or CHD4 by shRNA, or NuRD complex pharmacologic inhibition with the HDAC inhibitors such as Vorinostat resulted in significant cell growth arrest without cell death. Standard and Fucci reporter-based cell cycle analysis further confirmed that inhibition of the NuRD complex with Vorinostat induced 94.47% cells in the G0 phase (p<0.0001), suggesting a dense quiescent state. Through cell cycle phase maps produced by iterative immunofluorescence of 30 core cell cycle regulators and manifold learning, we found Vorinostat-treated OvCa cells demonstrated a completely different cell cycle structure from control cells, with most of the cells in an extreme G0 phase. Bulk RNA-seq of Vorinostat-treated cells in three OvCa cell lines validated the downregulation of pathways relating to cell cycle, cell division, and DNA replication (LFC<-1, p<0.05), which is consistent with stem cells. Upregulation of several pathways involved in proteostasis were also noted. We therefore performed a targeted drug screen for combinatorial therapy approaches to eradicate qOvCa. We found that Vorinostat combined with the proteasome inhibitor Carfilzomib demonstrated the most profound synergistic cell death (Loewe index synergy score 23-73). Increased proteasome activity was also found in both primary and Vorinostat-induced qOvCa, suggesting the essential role of the proteasome in qOvCa. Tumor xenograft studies confirmed that Vorinostat alone can delay tumor growth (p<0.05) and combinatorial therapy of Vorinostat and Carfilzomib is more efficacious (p=0.0049). Taken together, NuRD complex inhibition induces quiescence. Combination therapy with Vorinostat to induce quiescence combined with proteasome inhibitors resulted in the profound death of therapy-resistant quiescent cells. Our work suggests this is a novel therapeutic approach to eradicate chemoresistant qOvCa cells and increase cure rates. Citation Format: Qi Jiang, Michelle Ertel, Santiago Panesso- Gómez, Sara Sannino, April Sagan, Alexander J. Cole, Stacy C. McGonigal, Betsy Ann Varghese, Wayne Stallaert, Jeffrey L. Brodsky, Hatice U. Osmanbeyoglu, Ronald J. Buckanovich. A new combinatorial therapy that synergistically targets the NuRD complex and proteostasis to eradicate quiescent ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5906.

Abstract 704: Targeting hypoxic survival pathways to overcome radiotherapy-related treatment resistance

Abstract Tumor cells undergo biological adaptations in order to survive stress conditions, such as hypoxia and nutrient deprivation. These biological adaptations include the activation of hypoxic survival pathways that play an important role in acquiring radioresistance which becomes most challenging with treatment regimens using high doses of radiation/fraction (e.g., hypofractionated radiotherapy). Therefore, inhibiting key regulators within these pathways and thereby exploiting these survival mechanisms, gives rise to new potential drug targets for combined treatment modalities. This novel strategy investigated in our laboratory relies on the concept of biological cooperation: while ionizing radiation kills primarily well-oxygenated cells, such inhibitors of survival kinases will drive quiescent tumor cells under hypoxia and/or nutrient deprivation either directly into cell death or into re-entry into a proliferative and thereby more ionizing radiation-sensitive state. One of the survival kinases we are investigating in combination with ionizing radiation (IR) is DYRK1B (dual-specificity tyrosine regulated kinase 1B). We demonstrated that the expression of DYRK1B was upregulated under serum-starvation and hypoxia, but not in response to IR. The small molecule DYRK1B inhibitor AZ191 and shRNA-mediated DYRK1B knockdown significantly reduced proliferative activity and clonogenicity of SW620 tumor cells alone and in combination with IR under serum-starved conditions, which correlated with increased ROS levels and DNA damage. Furthermore, AZ191 successfully targeted the hypoxic core of tumor spheroids while IR preferentially targeted normoxic cells in the rim of the spheroids. A combined treatment effect was also observed in patient-derived colorectal carcinoma organoids but not in healthy tissue organoids. This data shows that inhibition of DYRK1B in quiescent tumor cells could drive tumor cells on their own into crisis or into a more radiosensitive cell cycle phase and thus supports our strategy of biological cooperation. In the continuation of this project, we have performed a drug-screen with approx. 3’000 clinically relevant compounds in colorectal tumor cells lines under normoxic and hypoxic conditions in order to explore additional hypoxia-mediated survival pathways contributing to radioresistance. Thereby we aim to identify novel combined treatment modalities to overcome radiotherapy-induced hypoxia and hypoxia-related resistance mechanisms. Citation Format: Claire Beckers, Lazaros Vasilikos, Alba Sanchez-Fernandez, Lorena Moor, Martin Pruschy. Targeting hypoxic survival pathways to overcome radiotherapy-related treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 704.

Abstract 5113: Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma

Abstract Radiation is one of the standard therapies for pediatric high-grade glioma (pHGG), of which the prognosis remains poor. To gain an in-depth understanding of the biological activities beyond the classic DNA damage, we treated 6 patient-derived orthotopic xenograft (PDOX) models, including one with DNA mismatch repair (MMR) deficiency, with fractionated radiations (2 Gy/day x 5 days). Extension of survival time was noted in 4/6 PDOX models (P<0.05) and widespread γH2AX positivity in >95% tumor cells in tumor core and >85% in the invasive foci, accompanied by ~30% apoptotic and mitotic catastrophic cell death. The model with DNA MMR (IC-1406HGG) was the most responsive to radiation with a reduction of Ki-67(+) cells. Altered metabolism, including mitochondria number elevation, COX IV activation and reactive oxygen species buildup, were detected together with the accumulation of CD133+ tumor cells. The latter was caused by the entry of quiescent G0 cells into cell cycle and the activation of self-renewal (SOX2 and BMI1) and epithelial mesenchymal transition (fibronectin) genes. These novel insights about the cellular and molecular mechanisms of fractionated radiation in vivo should support the development of new radio-sensitizing therapies. Citation Format: Zi-Lu Huang, Zhi-Gang Liu, Qi Lin, Ya-Lan Tao, Xinzhuoyun Li, Patricia Baxter, Jack MF Su, Adekunle M. Adesina, Chris Man, Murali Chintagumpala, Wan Yee Teo, Yu-Chen Du, Yun-Fei Xia, Xiao-Nan Li. Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5113.

Abstract 5443: Identifying key regulators of glioblastoma cancer stem cells

Abstract Glioblastoma multiforme (GBM) has a dire prognosis that has had little progress over the past three decades. The existence of a unique population of stem-like gene signature baring quiescent cells (GSCs) within GBM are important for GBM tumorigenesis, therapy-resistance and recurrence. Our lab has developed one of the most faithful genetically engineered mouse models, encompassing a Nestin promoter-enhancer driven CreERT2-H2BeGFP-hDTR cassette (hereafter as CGD), that allows us to accurately capture and isolate GSCs in mouse GBM. The next logical step is to leverage these tools to uncover therapeutic vulnerabilities of GSCs and make a meaningful impact on GBM patient outcomes. To our knowledge, very few studies have explored the functional regulators of GSCs using a CRISPR/Cas9 screening approach. Our GSC signature provides a strong foundation for performing targeted in vitro or in vivo screens. In this study, to identify functionally important genes that may regulate GSC self-renewal and tumorigenic properties, an in vitro CRISPR knockout screens using a gene library derived from the GSC signature. The viral library was applied to low passage primary cultures of FACS sorted CGD_GFP+ and CGD_GFP- GBM cells developed from CGD; Nf1f/+;Trp53f/f;Ptenf/+ mice, respectively. The result of our in vitro CRISPR knock-out screen demonstrated guide depletion of Atp1b2-sgRNA in CGD_GFP+ but not in CGD_GFP- cells. The main objective of this study is to utilize in vitro and in vivo functional assays to test the difference between Atp1b2 WT and Atp1b2 KO GSCs and Atp1b2 WT vs KO GSC-derived tumors. Having a targeted candidate gene list is also particularly helpful for designing an in vivo CRISPR knockout screen, which is more physiological relevant. Therefore, I propose to use the candidate gene list derived from the in vitro screen as a starting point. The in vivo screen can provide valuable insights for the selection of the most optimal candidates for anti-GSC therapies. Citation Format: Tao Wang, Xuanhua Xie, Luis F. Parada. Identifying key regulators of glioblastoma cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5443.