Knockout Cells Research Articles

CRISPR/Cas9-mediated RELA and RELC knockout in human regulatory T cells abrogates FOXP3 expression and suppressive function

Mutations in NF-κB-related molecules result in combined immunodeficiency characterized by recurrent infection. In this study, we aimed to investigate the association between mutations in NF-κB family members, RELA, RELB, and RELC, and regulatory T cell (Treg) function in humans. RELA, RELB, and RELC were knocked out using CRISPR/Cas9-mediated homologous recombination in CD4+/CD8+ T cells and Tregs isolated from healthy donors. The RELA, RELB, and RELC knockouts did not alter the phenotype or cytokine production profile of CD4+/CD8+ T cells or Jurkat cells. Similar to that observed in knockout mice, RELA or RELC knockout in MT-2 cells and freshly isolated Tregs reduced FOXP3 expression and the immune suppressive function of Tregs. Additionally, PD-L1 expression in effector T cells and Tregs decreased considerably following RELC knockdown. These findings demonstrated that the deletion of RELA or RELC resulted in the loss of Treg-like phenotype and function owing to the downregulation of FOXP3 expression.

Optimized lipid nanoparticles enable effective CRISPR/Cas9-mediated gene editing in dendritic cells for enhanced immunotherapy

Immunotherapy has emerged as a revolutionary approach to treat immune-related diseases. Dendritic cells (DCs) play a pivotal role in orchestrating immune responses, making them an attractive target for immunotherapeutic interventions. Modulation of gene expression in DCs using genome editing techniques, such as the CRISPR-Cas system, is important for regulating DC functions. However, the precise delivery of CRISPR-based therapies to DCs has posed a significant challenge. While lipid nanoparticles (LNPs) have been extensively studied for gene editing in tumor cells, their potential application in DCs has remained relatively unexplored. This study investigates the important role of cholesterol in regulating the efficiency of BAMEA-O16B lipid-assisted nanoparticles (BLANs) as carriers of CRISPR/Cas9 for gene editing in DCs. Remarkably, BLANs with low cholesterol density exhibit exceptional mRNA uptake, improved endosomal escape, and efficient single-guide RNA release capabilities. Administration of BLANmCas9/gPD-L1 results in substantial PD-L1 gene knockout in conventional dendritic cells (cDCs), accompanied by heightened cDC1 activation, T cell stimulation, and significant suppression of tumor growth. The study underscores the pivotal role of cholesterol density within LNPs, revealing potent influence on gene editing efficacy within DCs. This strategy holds immense promise for the field of cancer immunotherapy, offering a novel avenue for treating immune-related diseases.

NudCL2 is required for cytokinesis by stabilizing RCC2 with Hsp90 at the midbody.

Cytokinesis is required for faithful division of cytoplasmic components and duplicated nuclei into two daughter cells. Midbody, a protein-dense organelle that forms at the intercellular bridge, is indispensable for successful cytokinesis. However, the regulatory mechanism of cytokinesis at the midbody still remains elusive. Here, we unveil a critical role for NudC-like protein 2 (NudCL2), a co-chaperone of heat shock protein 90 (Hsp90), in cytokinesis regulation by stabilizing regulator of chromosome condensation 2 (RCC2) at the midbody in mammalian cells. NudCL2 localizes at the midbody, and its downregulation results in cytokinesis failure, multinucleation, and midbody disorganization. Using iTRAQ-based quantitative proteomic analysis, we find that RCC2 levels are decreased in NudCL2 knockout (KO) cells. Moreover, Hsp90 forms a complex with NudCL2 to stabilize RCC2, which is essential for cytokinesis. RCC2 depletion mirrors phenotypes observed in NudCL2-downregulated cells. Importantly, ectopic expression of RCC2 rescues the cytokinesis defects induced by NudCL2 deletion, but not vice versa. Together, our data reveal the significance of the NudCL2/Hsp90/RCC2 pathway in cytokinesis at the midbody.

The nuclear transportation of CHRONO regulates the circadian rhythm

The pace of the endogenous circadian clock is important for organisms to maintain homeostasis. CHRONO has been shown to be a core component of the mammalian clock and has recently been implicated to function in several important physiological aspects. To function properly, CHRONO needs to enter the nucleus to repress transcription. We have previously shown that the N-terminus of CHRONO is required for its nuclear entry. However, how CHRONO enters the nucleus and regulates the circadian clock remains unknown. Here, we report that a novel non-classical nuclear localization signal (NLS) in the N-terminus of CHRONO is responsible for its nuclear entry. Multiple nuclear transporters are identified that facilitate the nuclear import of CHRONO. We show that the Arg63 is the critical amino acid of the NLS. Using prime editing technology, we precisely edit the Arg63 to Ala at the genomic loci and demonstrate that this mutation prolongs the circadian period, which is similar to knockdown of CHRONO. By using the CHRONO knockout and R63A mutant cells, we also investigated the changes in the cytoplasmic/nuclear distribution of BMAL1. We show that BMAL1 localizes more in the cytoplasm in the deficiency of CHRONO nuclear entry. These results provide a model for CHRONO nuclear entry using a network of importins involved in the regulation of the circadian period.

HDAC7 promotes ovarian cancer malignancy via AKT/mTOR signalling pathway.

Ovarian cancer is of the most lethal malignancy and causes serious threat to women health worldwide. A deep understanding of molecular mechanisms underlying ovarian cancer progression is critical for the development of promising therapeutic strategies. In this study, we aimed to employ immunohistochemistry to determine the protein level of HDAC7 in patient tissues, our data showed HDAC7 levels are upregulated in tumour tissues. In addition, we also performed Kaplan-Meier survival analysis to investigate the association between HDAC7 expression and clinical prognosis, and found that HDAC7 expression was associated with poor prognosis in ovarian cancer patients. Inhibition of HDAC7 cells resulted in lower cell proliferation, invasion and colony formation. Furthermore, we also found that HDAC7 inhibition suppressed PI3K/AKT/mTOR pathway. In contrast, exogenous HDAC7 expression activated the PI3K/AKT/mTOR pathway in HDAC7 knockout cells and rescued the cell proliferation, invasion and colony formation. However, inhibition of p-AKT induced lower cell proliferation, metastasis and colony formation abilities. In murine model, HDAC7 KO significantly decreased the tumour burden. These data indicate that HDAC7 is involved in regulation of PI3K/AKT/mTOR pathway and targeting of HDAC7 could be potential therapeutic strategy in the treatment of ovarian cancer.

Multifunctional Dendrimer-Peptide Conjugates for MET Receptor-specific Imaging of Cancer Cells

The Mesenchymal Epithelial Transition (MET) receptor tyrosine kinase is frequently upregulated or mutated in various cancers. Targeting MET signaling pathway has been utilized as a treatment for cancer, as since MET overexpression is often associated with poor prognosis. Selective imaging of MET-overexpressing tumor cells would thus provide a high diagnostic value; however, it remains elusive due to a lack of targeted imaging contrast agents. Herein, we have developed a multifunctional diagnostic dendrimer-peptide conjugate (DPC) system with a strong avidity to MET-expressing cancer cells. The system was prepared by conjugating MET-inhibiting peptides (C7) to generation 7 (G7) poly(amidoamine) (PAMAM) dendrimers. Due to the dendrimer-mediated multivalent binding effect, the DPCs exhibited a significantly stronger binding to the human MET protein than free C7, as measured using surface plasmon resonance. Confocal microscopy revealed increased binding of the DPCs to the MET-expressing EBC-1 and UW-Lung-21 cells, whereas a MET knock-out cell line showed negligible interactions with the DPCs. The DPCs were then conjugated with Zirconium-89 for positron emission tomography and computed tomography (PET/CT) scanning, demonstrating their selective accumulation to MET-expressing tumors in vivo. Additionally, the plasma half-life of the DPCs was measured at ~53hours, which was significantly longer than free C7. These results collectively suggest that this DPC system has potential as a targeted imaging platform specific to MET-expressing tumors, which would be applicable to various cancer types.

Effects of ATG5 and ATG7 Knockout on Ferroptosis Sensitivity of RPMI-8226 Cells

To investigate the effects of ATG5 and ATG7 genes on the sensitivity of multiple myeloma (MM) cell line RPMI-8226 cells to ferroptosis. CRISPR/Cas9 technology was used to knock out the autophagy key genes ATG5 and ATG7 in RPMI-8226 cells. Western blot was used to identify gene knockout cells, and detect the expression changes of autophagy-related proteins P62 and LC3B. Flow cytometry was used to detect the change of sensitivity of gene knockout cells to RSL3. The content of intracellular ferrous ions and reactive oxygen species (ROS) level in gene knockout cells were detected. Western blot result confirmed that ATG5 and ATG7 genes were knocked out successfully in RPMI-8226 cells. The result of flow cytometry showed that the cell viability of RPMI-8226 cells was dose-dependent on different concentrations of RSL3 (r =-0.969). RSL3 (10 μmol/L) was used to induce ferroptosis in cells of control group and gene knockout groups, then the cell viability in gene knockout groups were both higher than control group after 48 hours (both P < 0.001). After knocking out the ATG5 and ATG7 genes, the content of intracellular Fe2+ decreased significantly compared with control group (both P < 0.01), and the ROS level also decreased (both P < 0.001). Knockout of ATG5 and ATG7 genes can inhibit the ferroptosis of MM cells, and LAP pathway may be involved in the regulation.

Modified (2′-deoxy)adenosines activate autophagy primarily through AMPK/ULK1-dependent pathway

Autophagy is a conserved self-digestion process, which governs regulated degradation of cellular components. Autophagy is upregulated upon energy shortage sensed by AMP-dependent protein kinase (AMPK). Autophagy activators might be contemplated as therapies for metabolic neurodegenerative diseases and obesity, as well as cancer, considering tumor-suppressive functions of autophagy. Among them, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr), a nucleoside precursor of the active phosphorylated AMP analog, is the most commonly used pharmacological modulator of AMPK activity, despite its multiple reported “off-target” effects. Here, we assessed the autophagy/mitophagy activation ability of a small set of (2′-deoxy)adenosine derivatives and analogs using a fluorescent reporter assay and immunoblotting analysis. The first two leader compounds, 7,8-dihydro-8-oxo-2′-deoxyadenosine and -adenosine, are nucleoside forms of major oxidative DNA and RNA lesions. The third, a derivative of inactive N6-methyladenosine with a metabolizable phosphate-masking group, exhibited the highest activity in the series. These compounds primarily contributed to the activation of AMPK and outperformed AICAr; however, retaining the activity in knockout cell lines for AMPK (ΔAMPK) and its upstream regulator SIRT1 (ΔSIRT1) suggests that AMPK is not a main cellular target. Overall, we confirmed the prospects of searching for autophagy activators among (2′-deoxy)adenosine derivatives and demonstrated the applicability of the phosphate-masking strategy for increasing their efficacy.

Effect of peroxiredoxin 1 or peroxiredoxin 2 knockout on the thiol proteome of Jurkat cells.

Peroxiredoxins are important regulators of cellular peroxide metabolism. As antioxidants, they restrict oxidation of other cell proteins, but as signaling molecules they can act as sensors and promote thiol protein oxidation via a redox relay mechanism. The presence of peroxiredoxins could therefore influence other thiol proteins, even in cells experiencing endogenous redox activity. To investigate this for the two cytoplasmic peroxiredoxins, Prdx1 and Prdx2, we have compared the thiol proteome of wildtype Jurkat cells with cells in which either one was knocked out. Using mass spectrometry and isotope tagging, approximately 10,000 common CysSH-containing peptides were detected for each WT/KO comparison. Knockout of Prdx1 or Prdx2 resulted in a change in redox state of a small selection of Cys residues, with less than 100 giving more than a 2-fold difference. Strikingly, a large proportion of these, including those that showed the greatest change, were common to both KOs. Some Cys residues showed more oxidation in the knockouts, whereas others showed less. The candidate proteins have diverse functions and have not been known to be oxidant sensitive. No differences were seen in redox state of Cys residues of other Prdxs and oxidant sensitive proteins. A change in expression in Prdx2 knockout cells was indicated for seven cytoskeletal or regulatory thiol proteins, three of which were tested and validated by western blotting. Little firm evidence was found for thiol redox changes dependent on either Prdx that could be attributed to oxidation via a relay mechanism.

Fatty acid synthase (FASN) is a tumor-cell-intrinsic metabolic checkpoint restricting T-cell immunity

Fatty acid synthase (FASN)-catalyzed endogenous lipogenesis is a hallmark of cancer metabolism. However, whether FASN is an intrinsic mechanism of tumor cell defense against T cell immunity remains unexplored. To test this hypothesis, here we combined bioinformatic analysis of the FASN-related immune cell landscape, real-time assessment of cell-based immunotherapy efficacy in CRISPR/Cas9-based FASN gene knockout (FASN KO) cell models, and mathematical and mechanistic evaluation of FASN-driven immunoresistance. FASN expression negatively correlates with infiltrating immune cells associated with cancer suppression, cytolytic activity signatures, and HLA-I expression. Cancer cells engineered to carry a loss-of-function mutation in FASN exhibit an enhanced cytolytic response and an accelerated extinction kinetics upon interaction with cytokine-activated T cells. Depletion of FASN results in reduced carrying capacity, accompanied by the suppression of mitochondrial OXPHOS and strong downregulation of electron transport chain complexes. Targeted FASN depletion primes cancer cells for mitochondrial apoptosis as it synergizes with BCL-2/BCL-XL-targeting BH3 mimetics to render cancer cells more susceptible to T-cell-mediated killing. FASN depletion prevents adaptive induction of PD-L1 in response to interferon-gamma and reduces constitutive overexpression of PD-L1 by abolishing PD-L1 post-translational palmitoylation. FASN is a novel tumor cell-intrinsic metabolic checkpoint that restricts T cell immunity and may be exploited to improve the efficacy of T cell-based immunotherapy.

Npac Regulates Pre-mRNA Splicing in Mouse Embryonic Stem Cells.

As a reader of tri-methylated lysine 36 on histone H3 (H3K36me3), Npac has been shown to have a significant role in gene transcription elongation. However, its potential implication in RNA splicing remains unknown. Here, we characterized the phenotypes of Npac knockout in mES cells. We discovered that loss of Npac disrupts pluripotency and identity in mESCs. We also found that Npac is associated with many cellular activities, including cell proliferation, differentiation, and transcription regulation. Notably, we uncovered that Npac is associated with RNA splicing machinery. Furthermore, we found that Npac regulates alternative splicing through its interaction with the splicing factors, including Srsf1. Our research thus highlights the important role of Npac in maintaining ESC identity through the regulation of pre-mRNA splicing.

A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.

RCC1 regulation of subcellular protein localization via Ran GTPase drives pancreatic ductal adenocarcinoma growth

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy, with limited therapeutic options. Here, we evaluated the role of regulator of chromosome condensation 1 (RCC1) in PDAC. RCC1 functions as a guanine exchange factor for GTP-binding nuclear protein Ran (Ran) GTPase and is involved in nucleocytoplasmic transport. RCC1 RNA expression is elevated in PDAC tissues compared to normal pancreatic tissues and correlates with poor prognosis. RCC1 silencing by RNAi and CRISPR-Cas9 knockout (KO) results in reduced proliferation in 2-D and 3-D cell cultures. RCC1 knockdown (KD) reduced migration and clonogenicity, enhanced apoptosis, and altered cell cycle progression in human PDAC and murine cells from LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) tumors. Mechanistically, RCC1 KO shows widespread transcriptomic alterations including regulation of PTK7, a co-receptor of the Wnt signaling pathway. RCC1 KD disrupted subcellular Ran localization and the Ran gradient. Nuclear and cytosolic proteomics revealed altered subcellular proteome localization in Rcc1 KD KPC-tumor-derived cells and several altered metabolic biosynthesis pathways. In vivo, RCC1 KO cells show reduced tumor growth potential when injected as sub-cutaneous xenografts. Finally, RCC1 KD sensitized PDAC cells to gemcitabine chemotherapy treatment. This study reveals the role of RCC1 in pancreatic cancer as a novel molecular vulnerability that could be exploited to enhance therapeutic response.

The identification of high-performing antibodies for FUS (Uniprot ID: P35637) for use in western blot, immunoprecipitation, immunofluorescence and flow cytometry

RNA-binding protein Fused-in Sarcoma (FUS) plays an essential role in various cellular processes. Mutations in the C-terminal domain region, where the nuclear localization signal (NLS) is located, causes the redistribution of FUS from the nucleus to the cytoplasm. In neurons, neurotoxic aggregates are formed as a result, contributing to neurogenerative diseases. Well-characterized anti-FUS antibodies would enable the reproducibility of FUS research, thereby benefiting the scientific community. In this study, we characterized ten FUS commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

LncRNA DCRT Protects Against Dilated Cardiomyopathy by Preventing NDUFS2 Alternative Splicing by Binding to PTBP1.

Dilated cardiomyopathy is characterized by left ventricular dilation and continuous systolic dysfunction. Mitochondrial impairment is critical in dilated cardiomyopathy; however, the underlying mechanisms remain unclear. Here, we explored the cardioprotective role of a heart-enriched long noncoding RNA, the dilated cardiomyopathy repressive transcript (DCRT), in maintaining mitochondrial function. The DCRT knockout (DCRT-/-) mice and DCRT knockout cells were developed using CRISPR-Cas9 technology. Cardiac-specific DCRT transgenic mice were generated using α-myosin heavy chain promoter. Chromatin coimmunoprecipitation, RNA immunoprecipitation, Western blot, and isoform sequencing were performed to investigate the underlying mechanisms. We found that the long noncoding RNA DCRT was highly enriched in the normal heart tissues and that its expression was significantly downregulated in the myocardium of patients with dilated cardiomyopathy. DCRT-/- mice spontaneously developed cardiac dysfunction and enlargement with mitochondrial impairment. DCRT transgene or overexpression with the recombinant adeno-associated virus system in mice attenuated cardiac dysfunction induced by transverse aortic constriction treatment. Mechanistically, DCRT inhibited the third exon skipping of NDUFS2 (NADH dehydrogenase ubiquinone iron-sulfur protein 2) by directly binding to PTBP1 (polypyrimidine tract binding protein 1) in the nucleus of cardiomyocytes. Skipping of the third exon of NDUFS2 induced mitochondrial dysfunction by competitively inhibiting mitochondrial complex I activity and binding to PRDX5 (peroxiredoxin 5) and suppressing its antioxidant activity. Furthermore, coenzyme Q10 partially alleviated mitochondrial dysfunction in cardiomyocytes caused by DCRT reduction. Our study revealed that the loss of DCRT contributed to PTBP1-mediated exon skipping of NDUFS2, thereby inducing cardiac mitochondrial dysfunction during dilated cardiomyopathy development, which could be partially treated with coenzyme Q10 supplementation.

Exploring the role of YBX3 in PEDV infection through the utilization of YBX3 knockout and overexpression cell lines.

Porcine epidemic diarrhea (PED) is a highly contagious disease caused by the porcine epidemic diarrhea virus (PEDV), which results in significant economic losses. PEDV infection causes severe damage to the midgut barrier in the small intestine. YBX3, an important protein in tight junctions, promotes epithelial cell proliferation. However, its role in the process of PEDV infection remains unclear. In this study, we observed a significant increase in mRNA expression of YBX3 following PEDV infection. Additionally, the protein expression of YBX3 showed an initial increase followed by a decrease over time. Furthermore, treatment with 2% DSS resulted in a significant down-regulation of YBX3 mRNA and protein expression. Furthermore, we successfully generated knockout and overexpression cell lines of YBX3. Preliminary assays indicated that elevated expression of YBX3 inhibited the PEDV replication, while knockout of YBX3 had the opposite effect. In conclusion, our study has preliminarily revealed the functional role of YBX3 during PEDV infection. This finding lays the foundation for further investigation into its mechanism in future and also provides new insights into the mechanism of PEDV-host interactions.

Mitochondrial membrane potential and oxidative stress interact to regulate Oma1-dependent processing of Opa1 and mitochondrial dynamics.

Mitochondrial form and function are regulated by the opposing forces of mitochondrial dynamics: fission and fusion. Mitochondrial dynamics are highly active and consequential during neuronal ischemia/reperfusion (I/R) injury. Mitochondrial fusion is executed at the mitochondrial inner membrane by Opa1. The balance of long (L-Opa1) and proteolytically cleaved short (S-Opa1) isoforms is critical for efficient fusion. Oma1 is the predominant stress-responsive protease for Opa1 processing. In neuronal cell models, we assessed Oma1 and Opa1 regulation during mitochondrial stress. In an immortalized mouse hippocampal neuron line (HT22), Oma1 was sensitive to mitochondrial membrane potential depolarization (rotenone, FCCP) and hyperpolarization (oligomycin). Further, oxidative stress was sufficient to increase Oma1 activity and necessary for depolarization-induced proteolysis. We generated Oma1 knockout (KO) HT22 cells that displayed normal mitochondrial morphology and fusion capabilities. FCCP-induced mitochondrial fragmentation was exacerbated in Oma1 KO cells. However, Oma1 KO cells were better equipped to perform restorative fusion after fragmentation, presumably due to preserved L-Opa1. We extended our investigations to a combinatorial stress of neuronal oxygen-glucose deprivation and reoxygenation (OGD/R), where we found that Opa1 processing and Oma1 activation were initiated during OGD in an ROS-dependent manner. These findings highlight a novel dependence of Oma1 on oxidative stress in response to depolarization. Further, we demonstrate contrasting fission/fusion roles for Oma1 in the acute response and recovery stages of mitochondrial stress. Collectively, our results add intersectionality and nuance to the previously proposed models of Oma1 activity.

Relationship between pathogenic E.coli O78-induced intestinal epithelial barrier damage and Zonulin expression levels in yaks.

The Zonulin gene overexpression and knockout cell lines were successfully constructed, the TEER value of the barrier of Zonulin overexpression cell lines began to decrease at 1 h after the addition of E.coli O78 and reached the lowest value at 4 h, and the TEER value of Zonulin interference cell lines decreased within 1-4 h after the addition of E.coli O78. At 4 h, the FD4 passing capacity of Zonulin overexpression cell lines was significantly higher than that of interfering cell lines, reaching twice as much as siRNA-1. The amount of bacterial translocation in overexpressed cell lines increased rapidly within 1-4 h, and the concentration of E.coli in the lower chamber was significantly higher than that in the siRNA-1 group at 4 h, but there was no significant change in the siRNA-1 group in the 1-4 h. There was no significant change in the mRNA level of MUC1 in Zonulin overexpression and interference cell lines after the addition of E.coli O78. In the overexpression group, the mRNA levels of MUC2, Occludin, and ZO-1 were significantly decreased, and the mRNA level of FABP2 was increased considerably. These results suggest stimulate epithelial cells to secrete Zonulin protein. Many Zonulin proteins regulate the opening of tight junction structures, reduce the transmembrane resistance of the cell barrier, and improve the permeability of the cell barrier and the amount of bacterial translocation.

Abstract C091: Uncovering genetic regulators of metabolic plasticity in renal cell carcinoma

Abstract We aim to offer new insights on the regulation of metabolic plasticity in renal cell carcinoma (RCC) and identify novel metabolic regulatory targets that can be used for potential treatment therapies of the disease. RCC affects an estimated 400,000 people worldwide each year with over 100,00 deaths annually that disproportionately affects racial/ethnic minorities in the United States. RCC is a term that covers an array of kidney cancer subtypes differing in incidence, histopathology, genetic and molecular alterations, as well as in clinical outcomes and prognoses. However, a common characteristic of many RCCs is that they are driven by metabolic rewiring, due to a high frequency of mutations in genes that regulate major metabolic processes in the cell. It was recently discovered that patient overall survival and prognosis has been linked to the expression of certain metabolic signatures in RCC tumor cells. Metabolic plasticity allows cancer cells to survive and adapt to different environments and metabolic stress conditions, such as nutrient deprivation. Therefore, exploring and exploiting metabolic rewiring in these cells may reveal genetic vulnerabilities and dependencies. This could lead to discovery of novel biomarkers and molecular targets that can be implicated as future therapeutic interventions for this disease. To this end, we applied functional genomic screening to expose genetic dependencies and vulnerabilities under different physiologic and metabolic conditions in patient derived medullary (PDM) RCC cells. The medullary RCC is a rare and aggressive RCC subtype that disproportionately affects the African American/Black population. We applied a CRISPR- based combinatorial screening platform that is based on co-expression of Cas9 and Cas12a nucleases and libraries of hybrid guide RNAs (hgRNA) to achieve ultra-efficient gene knockout (Aregger et al., 2021; Gonatopoulos-Pournatzis et al., 2020) in PDM RCC cells. Additionally, cells were screened under different media conditions that mimic human physiological nutrient conditions and a media deprived of lipids, one of the major metabolic substrates of the cell. By utilizing a genome-wide pooled CRISPR gene knockout screen we were able to systematically uncover genes required for cell survival and proliferation in a panel of patient-derived RCCs. We have begun to uncover genetic dependencies and major metabolic pathways that can be elucidated and exploited for future studies, such as those involving ferroptosis which is a targetable pathway for the development of cancer treatment therapies. Our findings demonstrate some of the key genetic drivers underlying cellular adaptations to different physiological environments and nutrient deprivation. In future studies we will further explore the relationship between different key metabolic regulatory genes in RCC and map for genetic interactions in major metabolic pathways. Citation Format: Chelsee Holloway, Josef Horak, Youngkyu Jeon, Michael Aregger. Uncovering genetic regulators of metabolic plasticity in renal cell carcinoma [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C091.

Identification of Cables1 as a critical host factor that promotes ALV-J replication via genome-wide CRISPR/Cas9 gene knockout screening

Avian leukosis virus subgroup J (ALV-J), a member of the genus Alpharetrovirus, possesses a small genome and exploits a vast array of host factors during its replication cycle. To identify host factors required for ALV-J replication and potentially guide the development of key therapeutic targets for ALV-J prevention, we employed a chicken genome-wide CRISPR/Cas9 knockout library to screen host factors involved in ALV-J infection within DF-1 cells. This screening revealed 42 host factors critical for ALV-J infection. Subsequent knockout assays showed that the absence of the genes encoding cycle-regulatory proteins, namely Cables1, CDK1, and DHFR, significantly inhibited ALV-J replication. Notably, Cables1 knockout cell lines displayed the most pronounced inhibitory effect. Conversely, overexpression assays confirmed that Cables1 significantly promotes ALV-J replication. Immunoprecipitation assays further indicated that Cables1 specifically interacts with the viral protein p15 (viral protease) among all ALV-J proteins, enhancing ALV-J p15 polyubiquitination. Additionally, we identified 26 lysine residues of ALV-J p15 as key sites for ubiquitination, and their replacement with arginine attenuated the replication ability of ALV-J in both in vitro and in vivo assays. This study demonstrates that Cables1 is a critical replication-dependent host factor of ALV-J by enhancing p15 ubiquitination and thereby promoting viral replication. Overall, these findings contribute to a deeper understanding of the ALJ-V replication mechanism and offer a potential target for the prevention and control of ALV-J infection.