Single-Cell Transcriptomics of Human TET2 Knockout CD4 T-Cells and Their Clonal Evolution

Progenitor cells at the basal layer of skin epidermis play an essential role in maintaining tissue homeostasis and enhancing wound repair in skin. The proliferation, differentiation, and cell death of epidermal progenitor cells have to be delicately regulated, as deregulation of this process can lead to many skin diseases, including skin cancers. However, the underlying molecular mechanisms involved in skin homeostasis remain poorly defined. In this study, with quantitative proteomics approach, we identified an important interaction between KDF1 (keratinocyte differentiation factor 1) and IKKα (IκB kinase α) in differentiating skin keratinocytes. Ablation of either KDF1 or IKKα in mice leads to similar but striking abnormalities in skin development, particularly in skin epidermal differentiation. With biochemical and mouse genetics approach, we further demonstrate that the interaction of IKKα and KDF1 is essential for epidermal differentiation. To probe deeper into the mechanisms, we find that KDF1 associates with a deubiquitinating protease USP7 (ubiquitin-specific peptidase 7), and KDF1 can regulate skin differentiation through deubiquitination and stabilization of IKKα. Taken together, our study unravels an important molecular mechanism underlying epidermal differentiation and skin tissue homeostasis.

Role and Mechanisms of Rhoa G17V in the Pathogenesis of AITL

e17544 Background: Epithelial to Mesenchymal Transition (EMT) is essential to the process of metastasis in ovarian cancer (OC). Early expressions of proteins and pathways during EMT essential for understanding mechanism and potential therapeutic targets in OC. The aim of our study is to evaluate the role and mechanism of Connective Tissue Growth Factor (CTGF) in OC. Methods: R182 and R2615 are well-characterized epithelial OC cell lines. CTGF expression +/- Transforming Growth Factor -b (TGF-b) was determined via Western blot. R182 and R2615 CTGF knock out (KO) were derived utilizing a Cas9/CRISPR-Cas9 lentivirus plasmid vector and confirmed via PCR. Anoikis resistance and invasion assays were performed to characterize phenotypes of R182 and R2615 wild type (WT) and KO cells. For anoikis resistance, cells were plated in triplicate in an ultra-low adhesive (ULA) cell plate Promega CellTiter assay which measured cell viability quantified by absorbance at 450 nm at 0, 24, 48, and 72 hrs. For invasion assay, 3000 cells were suspended in 50% reduced growth factor Basement Membrane Extract. Human recombinant CTGF was added at 50 and 100 ng/mL concentrations. Cells were plated in tissue culture plate and placed in Cytation 5/Biospa and imaged at 4-hour interval for up to 6 days. Western blot evaluated expression of mesenchymal markers. Chemo-sensitivity was measured by half maximal inhibitory concentration (IC50) for Cisplatin between the WT and KO cells. RNA sequence analysis was performed for both R182 WT and KO cells and analyzed utilizing iPathway Guide. Results: CTGF is constitutively expressed in R182 and R2615 OC cells line as early as 6 hrs of culture and after 30 min of TGF-b treatment. Loss of CTGF promoted anoikis resistance. At 72 hr, R182 CTGF WT cells displayed 75% viability while R182 KO only have 10% viability. Further, we demonstrated that loss of CTGF increases invasion. Administration of exogeneous CTGF in KO cells suppresses invasion in a dose dependent manner demonstrated. Along with epithelial markers CK-18 and beta-catenin, the KO cells expressed the mesenchymal marker SNAIL. No difference in chemo-sensitivity to Cisplatin was seen between WT and KO in either cell line. RNA sequence analysis identified 1106 out of 14054 differentially expressed genes between the WT and KO cells. PI3KAkt, cell adhesion molecules and ECM receptor interaction were pathway that were most significantly regulated. Conclusions: CTGF expression maintains the epithelial phenotype of OC cells during EMT. Loss of CTGF allows anoikis resistance and invasion which are vital characteristics in the metastatic nature of OC. We suggest that loss of CTGF expression in OC cells could be a biomarker for more aggressive OC.

Cells of the artery wall may accumulate free cholesterol and cholesteryl esters becoming foam cells. Up to 50% of foam cells in human lesions originates from smooth muscle cells (SMCs). Arterial SMCs express the ATP binding cassette (ABC) transporter ABCA1 and, upon cholesterol loading, express macrophage markers and a phagocytic activity. To characterize the role of ABCA1 and HDL3 in this transdifferentiation process, we evaluated the phenotypic changes in SMCs isolated from wild type (WT) and ABCA1 knock out (KO) mice and how HDL3 affects these changes. Cholesterol loading causes the downregulation of the expression of SMC markers including ACTA2, alpha-tropomyosin and myosin heavy chain and increases the expression of macrophage-related genes such as CD68, Mac-2, SRB1, MMPs, ABCG1 and ABCA1. HDL3 treatment in WT cells is able to normalize the expression of ACTA2, while the expression of macrophage-related genes is reduced. On the contrary, the preventive effect of HDL3 is completely lost in ABCA1 KO cells. Interestingly, the presence of HDL3 does not differently affect neutral lipid accumulation in WT or ABCA1 KO cells but stimulates phospholipids removal only in WT cells. ApoAI addition does not reverse the phenotypic changes induced by cholesterol not only in KO but also in WT cells. Moreover, cholesterol loading reduces the expression of myocardin, the master SMC specific-transcriptional coactivator involved in SMC differentiation, by up to 55% (p<0.01 vs respective control) in both cell types. HDL3 normalizes myocardin levels in WT cells while it does not have any effect in ABCA1 KO cells. Similar results are obtained evaluating the levels of miR-143/145, which positively regulate myocardin. The basal expression level of KLF4, a myocardin repressor, is almost double in ABCA1 KO cells compared to WT. After cholesterol loading, KLF4 is slightly reduced in WT cells, while its expression is halved in ABCA1 KO cells. HDL3 restores KLF4 to basal levels in KO cells, but it further reduces them in WT cells. These results indicate that HDL3, modulating the miR143/145-myocardin axis in SMC, prevents the cholesterol-induced gene expression modification regardless of its cholesterol unloading capacity and the presence of ABCA1 is required.

In humans, AMPylation of cellular proteins is carried out by Huntingtin yeast-interacting protein E (HYPE), activated under conditions of endoplasmic reticulum stress, such as in cancer cells. Extracts of the human chronic lymphocytic leukemia cell line, OSU-CLL, were fractionated using immuno-precipitation with antibodies against adenosine-phosphate and then AMP-Tyr. The proteins isolated were modified with AMP, the ‘AMPylome.’ AMP-labelled peptides isolated from HYPE wild-type (WT) and HYPE knock-out (KO) cells were identified using tandem mass spectrometry. A total of 213 proteins were identified from WT cell extracts, while only 23 of these were pulled down from KO cells, consistent with the presence of another AMPylator, besides HYPE. The KO cells were more sensitive to fludarabine nucleoside (2-FaraA) than WT cells. Ingenuity Pathway Analysis of the AMPylated proteins identified in WT cells clustered actin binding proteins of the cytoskeleton, and proteins of the RHO GTPase pathway that would jointly stimulate cell proliferation.

Many cell surface proteins are anchored to the membrane via a glycosylphosphatidylinositol (GPI) moiety, which is attached to the C terminus of the proteins. The core of the GPI anchor is conserved in all eukaryotes but is modified by various side chains. We cloned a mouse phosphatidylinositol glycan-class N (Pig-n) gene that encodes a 931amino acid protein expressed in the endoplasmic reticulum, which is homologous to yeast Mcd4p. We disrupted the gene in F9 embryonal carcinoma cells. In the Pig-n knockout cells, the first mannose in the GPI precursors was not modified by phosphoethanolamine. Nevertheless, further biosynthetic steps continued with the addition of the third mannose and the terminal phosphoethanolamine. The surface expression of Thy-1 was only partially affected, indicating that modification of the first mannose by phosphoethanolamine is not essential for attachment of GPI anchors in mammalian cells. An inhibitor of GPI biosynthesis, YW3548/BE49385A, inhibited transfer of phosphoethanolamine to the first mannose in mammalian cells but only slightly affected the surface expression of GPI-anchored proteins. Biosynthesis of GPI in the Pig-n knockout cells was not affected by YW3548/BE49385A, and yeast overexpressing MCD4 was highly resistant to YW3548/BE49385A, suggesting that Pig-n and Mcd4p are targets of this drug.

Purpose/Objective(s): Pancreatic ductal adenocarcinoma (PDAC) is characterized by an unusually high degree of hypoxia in the tumor microenvironment, which activates hypoxic signaling pathways orchestrated by hypoxia-inducible factor 1α (HIF1α). It has been shown previously that HIF1α significantly increases PDAC radioresistance and growth, though its mechanism of action is unknown. Here, we test the hypothesis that HIF1α confers these traits in PDAC through involvement in DNA damage repair and KRAS signaling in vitro. Materials/Methods: We performed studies with the KPC cell line (KrasLSL-G12D/+;TP53LSL-R172H/+). We used CRISPR-Cas9, a genome editing tool, to generate a HIF1α knockout (KO) cell line. Hypoxic microenvironments were induced using 100uM of the oxygen scavenger CoCl2. Radioresistance and double-strand DNA damage was evaluated and compared between wild-type (WT) and KO cells after treatment with increasing doses of ionizing radiation using neutral comet assays. Tumor signaling pathways were mapped and analyzed using western blot and co-immunoprecipitation. The KRASG12D inhibitor, BI-2852, was obtained from Boehringer-Ingelheim. Cancer cells were evaluated for growth and migration using cell proliferation assays and scratch wound assays following treatment with 50 uM BI-2852. Bioinformatic analysis was conducted using TCGA (The Cancer Genome Atlas) data; high and low expression were defined as samples with mRNA expression z-scores ≥ 1 and ≤ -1, respectively, Results: HIF1α KO cells demonstrated significantly increased radioresistance compared to WT cells following 8 and 10 Gy of radiation in hypoxic conditions (t-test, P<0.05). The olive moment of KO cells treated with the IC50 radiation dose (4 Gy) was significantly higher than that of WT cells (t-test, P<0.0001), suggesting that the DNA damage repair was upregulated by HIF1α. In addition, WT cells demonstrated higher tumor proliferation (t-test, P<0.01) and migration (t-test, P<0.05) compared to KO cells under hypoxia, suggesting HIF1α promoted tumor growth. WT cells treated with BI-2852 demonstrated significantly lower levels of TP53; using coimmunoprecipitation, we show that TP53 was likely degraded through Mdm2. HIF1α KO was necessary to induce high levels of apoptosis in cells treated with BI-2852. Our results suggest that HIF1α may act synergistically with KRAS to promote tumor survival through anti-apoptotic pathways that target TP53 for degradation. TCGA data also suggest that tumors with high expression of HIF1α is are associated with significantly poorer prognosis coimpared to tumors with low expression of HIF1α (Logrank test, P=0.003). Conclusion: Overall, this study demonstrates that HIF1α promotes DNA damage repair in PDAC and reveals a HIF1α/KRAS/p53 signaling axis. Future studies will test anti-HIF1α and anti-KRAS combination therapies in PDAC mouse models. Citation Format: Kevin J. Tu, Sanjit K. Roy, Amit Sawant, Hem D. Shukla. HIF1α is involved in radiation-induced DNA damage repair and anti-apoptotic pathways in pancreatic ductal adenocarcinoma. [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 4810.

Bcl‐2‐associated athanogene 3 (BAG3) is the only member of the BAG cochaperone family that is induced by stressful stimuli such as heat shock and heavy metals. In the present study, bag3 knockout (KO) HeLa cells were generated via the CRISPR‐Cas9 system, and the role of BAG3 in relation to p53 under heat stress conditions was investigated. Normally, the levels of p53 were low in both wild‐type (WT) and KO cells, while heat shock increased the levels of nuclear p53 in both cell lines. However, the increased level of p53 was much greater in WT cells than in KO cells, which suggested that BAG3 played a role in controlling the level of p53 under heat stress conditions. The mRNA level of p53 did not increase in either WT or KO cells during the heat stress period, which suggested that the differences in the levels of p53 were not due to transcriptional regulation. Both treatment with the proteasome inhibitor MG132 and heat shock drastically increased p53 levels to a similar extent in WT cells. Interestingly, both BAG3 and Hsp70 rapidly translocated to the nucleus and formed a complex with p53 upon heat stress. During a 1‐h recovery period from heat stress, the transcriptional activity of p53 increased up to 4‐fold in WT cells, but only 1.69‐fold in KO cells. These results demonstrate that Hsp70 and BAG3 are involved in the quality control of p53 under heat stress conditions and suggest a role for BAG3 as a cochaperone protein.

IntroductionWhile cyclin-dependent kinases (CDKs) have a key role in promoting/controlling transition between the different phases of the cell cycle, transcriptional kinases, like CDK12, are mainly involved in gene transcription. CDK12 has been shown to regulate the expression of genes involved in DNA damage and to maintain genomic stability. Impairment of CDK12 activity is synergic with PARP inhibitor and cisplatin treatments in different cellular systems. We here aimed to generate ovarian cancer cell lines knocked out (KO) for CDK12 to understand its role in ovarian cancer and in response to chemotherapy.Material and methodsA2780 and SKOV3 CDK12 KO clones were generated with CRISPR/Cas9 technology. Cell cycle analysis was evaluated by standard flow cytometric methods and DNA repair genes levels by Real Time PCR. Caspase 3 activity was measured to detect apoptosis with a luminescence-based assay. Cytotoxicity experiments were performed treating cells with different drug concentrations and evaluating cell survival after 72 hours by MTS assay. For in vivo studies 7.5 millions of cells were transplanted subcutaneously in nude mice and animals were monitored for tumour appearance and growth.Results and discussionsWe obtained 2 CDK12 KO ovarian cancer clones, A2780 KO and SKOV3 KO, out of more than 300 clones screened. The cell growth of both A2780 KO and SKOV3 KO cells is slower than the wild type (WT) cells, they have a less clonogenic ability and a tetraploid DNA content. Both CDK12 KO clones have a higher basal caspase activity than the WT cell lines, indicative of higher basal induction of apoptosis, while no increase in autophagy or senescence is observed. Both CDK12 KO clones show a decreased expression in BRCA1 and FANCD2 DNA repair genes than the WT cells. Cytotoxic experiments with anticancer agents with different mechanism of action show that both KO clones are less sensitive to ATM, CHK1 and WEE1 inhibitors treatment as compared to WT cells, while platinum and PARP inhibitors show similar cytotoxic activity in KO and WT cells. Interestingly enough, when KO clones were transplanted in nude mice, no tumour take was observed.ConclusionWe were able to obtain CDK12 KO cells. We think that these models could help in disclosing new roles of CDK12 in ovarian carcinoma and may represent a useful tool to study new combination therapies for tumours with CDK12 mutations.

PO-044 Possible involvement of G2/M block defects in cell sensitivity towards ATR inhibitors

PRMT3 is a type I arginine methyltransferase that resides in the cytoplasm. A large proportion of this cystosolic PRMT3 is found associated with ribosomes. It is tethered to the ribosomes through its interaction with rpS2, which is also its substrate. Here we show that mouse embryos with a targeted disruption of PRMT3 are small in size but survive after birth and attain a normal size in adulthood, thus displaying Minute-like characteristics. The ribosome protein rpS2 is hypomethylated in the absence of PRMT3, demonstrating that it is a bona fide, in vivo PRMT3 substrate that cannot be modified by other PRMTs. Finally, the levels 40 S, 60 S, and 80 S monosomes and polyribosomes are unaffected by the loss of PRMT3, but there are additional as yet unidentified proteins that co-fractionate with ribosomes that are also dedicated PRMT3 substrates.

A Multicenter, Open-Label, Phase 3, Randomized Controlled Trial of Duvelisib Versus Investigator's Choice of Gemcitabine or Bendamustine in Patients with Relapsed/Refractory Nodal T Cell Lymphoma with T Follicular Helper Phenotype

Dopamine D1 receptors (D1R), a subclass of G protein‐coupled receptors (GPCRs), regulate important neurobiological processes including movement, memory and responses to rewarding stimuli. D1Rs are also drug targets for treating motor deficits in Parkinson's disease and cognitive and motivational deficits in neuropsychiatric diseases. The D1R canonically activates heterotrimeric Gs and Golf G‐proteins which, in turn, activate adenylyl cyclase to increase cAMP/PKA signaling; however, the D1R also engages β‐arrestin proteins which may transduce an independent wave of intracellular signal transduction by scaffolding and activating kinases. The relative importance of G proteins versus arrestins for controlling the efficacy and duration of D1R signaling remains largely undefined. Here we delineate transducer‐specific contributions involved in D1R signaling using CRISPR/Cas9 genome editing to stably knockout (KO) Gs, Golf, multiple G‐proteins (Gs, Golf, Gq, G11 and G12) and β‐arrestin1/2 in HEK293 cells. Western blots confirmed a complete loss of the various G proteins or β‐arrestin1/2 proteins in KO cells versus wildtype cells. By measuring kinetic live‐cell cAMP production using the GloSensor and dose responses of selective agonists for the D1R (A‐77636, SKF‐81297), β2AR (isoproterenol) or Gs/olf (cholera toxin), we confirmed no cAMP was generated in the Gs/olf and multiple G protein KO cells. In contrast, β‐arrestin 1/2 KO greatly elevated and prolonged the duration of cAMP production after time course treatments with agonists for the D1R or the β2AR. Using antibody feeding followed by live‐cell confocal imaging of an HA tagged D1R, or a cell surface ELISA assay, we determined agonist‐mediated HA‐D1R endocytosis was entirely blocked in β‐arrestin 1/2 KO cells, but that Gs/olf KO had no effect on endocytosis. β‐arrestin1/2 KO also showed impaired agonist‐mediated D1R desensitization. Re‐expression of β‐arrestin 1 or 2 or both isoforms together in KO cells, restored receptor endocytosis and reduced the elevated D1R cAMP responses to that of wildtype cells. Lastly, knockout of Gs/olf or β‐arrestin1/2 differentially influenced the activation level and duration of ERK phosphorylation (p‐ERK). Western blotting indicated Gs/olf knockout completely blocked agonist‐induced p‐ERK induction by both D1R and β2AR agonists over a time course of 40 minutes. However, β‐arrestin1/2 knockout greatly increased and prolonged the duration of D1R and β2AR‐mediated p‐ERK. Taken together, this study provides fundamental insights into mechanisms of D1R signal transduction suggesting β‐arrestins are essential for D1R endocytosis and they primarily limit the intensity and duration of Gs/olf‐mediated D1R signaling. Gs/olf was essential for activating both cAMP signaling and ERK phosphorylation. Using this robust CRISPR/Cas9 KO model, future studies will examine downstream signaling events using proteomics and biased agonists to further define the role of both β‐arrestins and Gs/Golf for D1R pharmacology and signaling.Support or Funding InformationUTMB Center for Addiction Research and Dept. of Pharmacology and ToxicologyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Osteoclasts and foreign body giant cells (FBGCs) are multi-nuclear cells established by fusion of their mono-nuclear forms. Multi-nucleation via cell-cell fusion is a common characteristic of osteoclasts and FBGCs, and Dendritic Cell-Specific Transmembrane Protein (DC-STAMP) and Osteoclast Stimulatory Transmembrane Protein (OC-STAMP) are both required for the process. Thus, it is thought that DC-STAMP and OC-STAMP interaction likely induces this fusion, but details of these mechanisms are not clear. We crossed DC-STAMP knockout (KO) with OC-STAMP KO mice to obtain DC-STAMP/OC-STAMP doubly deficient (DKO) mice. Osteoclasts and FBGC common progenitors were isolated from wild-type (WT), DC-STAMP KO, OC-STAMP KO or DKO mice. We then set up 4 co-culture systems: (1) WT with DC-STAMP KO cells, (2) WT with OC-STAMP KO cells, (3) WT with DKO cells, and (4) DC-STAMP KO with OC-STAMP KO cells to induce osteoclast or FBGC formation. We evaluated osteoclast and FBGC formation by TRAP and May-Gruenwald Giemsa staining, respectively. Finally, we performed bone morphometric analysis of WT, DC-STAMP KO, OC-STAMP KO, and DKO mice. Cell-cell fusion occurs normally in co-cultures of DC KO with WT cells, OC KO with WT cells, and DKO with WT cells in both osteoclast and FBGC-inducing conditions. By contrast, co-cultures of DC-STAMP KO with OC-STAMP KO cells did not show cell-cell fusion. Bone morphometric analysis revealed enhanced bone formation in DKO mice. DC-STAMP and OC-STAMP cooperate to promote osteoclast and FBGC cell-cell fusion. DC-STAMP and OC-STAMP doubly deficient mice exhibit increased osteogenesis.

In breast cancer, steroid receptor coactivator-1 (SRC-1) expression positively correlates with HER2 expression and poor prognosis. In mouse mammary tumor virus-polyoma middle T (PyMT) breast cancer mouse model, SRC-1 strongly promotes mammary tumor metastasis. However, the molecular targets and mechanisms that mediate the role of SRC-1 in metastasis are unknown. In this study, SRC-1 wild-type (WT) and knockout (KO) cell lines were developed from the mammary tumors of WT/PyMT and KO/PyMT mice. WT cells exhibited strong migration and invasion capabilities, reduced E-cadherin and beta-catenin epithelial markers, gained N-cadherin and vimentin mesenchymal markers, and formed undifferentiated invasive structures in three-dimensional culture. In contrast, KO cells showed slow migration and invasion, retained E-cadherin, had less N-cadherin and vimentin, and developed partially differentiated three-dimensional structures. Importantly, WT cells expressed Twist, a master regulator of metastasis, at significantly higher levels versus KO cells. SRC-1 knockdown in WT cells reduced Twist expression, whereas SRC-1 restoration in KO cells also rescued Twist expression. Furthermore, SRC-1 was found to coactivate Twist transcription through physical interaction with the transcription factor PEA3 at the proximal Twist promoter. Accordingly, Twist knockdown in WT cells increased E-cadherin and reduced cell invasion and metastasis, and Twist expression in KO cells decreased E-cadherin and increased cell invasion. SRC-1 knockdown in human breast cancer cells also decreased Twist, cell migration, and invasion. Therefore, SRC-1 promotes breast cancer invasiveness and metastasis by coactivating PEA3-mediated Twist expression. Intervention of SRC-1 function may provide new strategies to inhibit breast cancer metastasis.