Null Line Research Articles

Multi-endpoint analysis of cadmium chloride-induced genotoxicity shows role for reactive oxygen species and p53 activation in DNA damage induction, cell cycle irregularities, and cell size aberrations.

Cadmium chloride (CdCl2) is a known genotoxic carcinogen, with a mechanism of action thought to partly involve the generation of reactive oxygen species (ROS). We applied here a multi-endpoint approach in vitro to explore the impact of CdCl2 on both the genome and on wider cell biology pathways relevant to cancer. Multi-endpoint approaches are believed to offer greater promise in terms of understanding the holistic effects of carcinogens in vitro. This richer understanding may help better classification of carcinogens as well as allowing detailed mechanisms of action to be identified. We found that CdCl2 caused DNA damage [micronuclei (MN)] in both TK6 and NH32 cells in a dose-dependent manner after 4 h exposure (plus 23 h recovery), with lowest observable effect levels (LOELs) for MN induction of 1 μM (TK6) and 1.6 μM (NH32). This DNA damage induction in TK6 cells was ROS dependent as pretreatment with the antioxidant N-Acetyl Cysteine (1 mM), abrogated this effect. However, 2',7'-dichlorofluorescin diacetate was not capable of detecting the ROS induced by CdCl2. The use of NH32 cells allowed an investigation of the role of p53 as they are a p53 null cell line derived from TK6. NH32 showed a 10-fold increase in MN in untreated cells and a similar dose-dependent effect after CdCl2 treatment. In TK6 cells, CdCl2 also caused activation of p53 (accumulation of total and phosphorylated p53), imposition of cell cycle checkpoints (G2/M) and intriguingly the production of smaller and more eccentric (elongated) cells. Overall, this multi-endpoint study suggests a carcinogenic mechanism of CdCl2 involving ROS generation, oxidative DNA damage and p53 activation, leading to cell cycle abnormalities and impacts of cell size and shape. This study shows how the integration of multiple cell biology endpoints studied in parallel in vitro can help mechanistic understanding of how carcinogens disrupt normal cell biology.

Loss of TP53 cooperates with c-MET overexpression to drive hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is a deadly malignancy with high genetic heterogeneity. TP53 mutation and c-MET activation are frequent events in human HCCs. Here, we discovered that the simultaneous mutations in TP53 and activation of c-MET occur in ~20% of human HCCs, and these patients show a poor prognosis. Importantly, we found that concomitant deletion of Trp53 and overexpression of c-MET (c-MET/sgp53) in the mouse liver led to HCC formation in vivo. Consistent with human HCCs, RNAseq showed that c-MET/sgp53 mouse HCCs were characterized by activated c-MET and Ras/MAPK cascades and increased tumor cell proliferation. Subsequently, a stably passaged cell line derived from a c-MET/sgp53 HCC and corresponding subcutaneous xenografts were generated. Also, in silico analysis suggested that the MEK inhibitor trametinib has a higher inhibition score in TP53 null human HCC cell lines, which was validated experimentally. We consistently found that trametinib effectively inhibited the growth of c-MET/sgp53 HCC cells and xenografts, supporting the possible usefulness of this drug for treating human HCCs with TP53-null mutations. Altogether, our study demonstrates that loss of TP53 cooperates with c-MET to drive hepatocarcinogenesis in vivo. The c-MET/sgp53 mouse model and derived HCC cell lines represent novel and useful preclinical tools to study hepatocarcinogenesis in the TP53 null background.

PINOID andPIN-FORMED Paralogous Genes Are Required forLeaf Morphogenesis inRice.

Auxin plays an essential role in modulating leaf development. However, its role in leaf development in rice (Oryza sativa L.) remains largely unknown. In this study, we found that PINOID (OsPID) and two Sister-of-PIN1s, termed PIN-FORMED1c (OsPIN1c) and OsPIN1d, are necessary for rice leaf development. The ospin1c ospin1d null mutant lines presented severe defects in leaf morphogenesis, including drooping and semi-drooping blades, an abnormally thickened sheath and lamina joint, and fused leaves with absent ligules and auricles. Loss-of-function ospid mutants displayed generally similar leaf morphology but lacked leaf fusion. Interestingly, misshaped leaf genesis displayed a preference for being ipsilateral. In addition, OsPIN1c and OsPID were commonly localized in the initiating leaf primordia. Furthermore, accompanied by the more severe organ morphogenesis in the ospin1c ospin1d ospid triple mutant, RNA sequencing analysis revealed that many genes essential for leaf development have an altered expression level. Together, this study furthers our understanding of the role auxin transport plays during leaf development in monocot rice.

BIOL-13. DEPLOYING NEW MODELS OF NF-1 MUTANT LOW GRADE GLIOMA TO ACCELERATE THERAPEUTIC DEVELOPMENT

Abstract Pediatric low grade glioma (pLGG) research is hampered by a paucity of genetically accurate cell line models and xenografts. To address this need, we have developed and characterized multiple in vivo and organoid models. We used cell reprogramming conditions (CRC) to establish and propagate JHH-NF1-PA1, a NF1-mutant line derived from a low grade optic pathway glioma of a patient with type 1 neurofibromatosis. JHH-NF1-PA1 cells persist and take up midline positions when xenografted into the brains of zebrafish, allowing testing of therapeutics in vivo. We developed a human iPSC-brain organoid system comprising of midline neurons, glia, and microglia, largely recapitulating the microenvironment of midline low grade glioma. Our JHH-NF1-PA1 cells incorporate into these brain organoids, allowing us to test potential therapeutics in an in vitro setting in which the tumor cells grow in the appropriate microenvironment. We have also further defined the mouse BL6 NF1 null glioma cell line 1810, which forms orthotopic xenografts in mice. This last model will be useful for therapeutic testing in a syngeneic BL6 background, allowing assessment of immune infiltration and signaling downstream of NF1 loss and potential immunotherapeutics. Together, these in vitro and in vivo models of NF1-driven low grade glioma make an important contribution to our understanding of the biology of low grade glioma.

CRISPR-Cas9-mediated disruption of B2M and CIITA genes eliminates HLA class I and II expression in human induced pluripotent stem cells (MUSIi001-A-2)

Cell-based therapy offers great promise for treating degenerative diseases. Although autologous cell-based therapy is ideal, it may be impractical due to the high manufacturing cost and long production time. Allogeneic cell-based therapy offers a more cost-effective alternative; however, the risk of graft rejection is a major concern. Here, we generated HLA class-I and -II null iPSC line by knocking out CIITA gene in the B2M-knockout MUSIi001-A-1 cell line using CRISPR/Cas9 system. The MUSIi001-A-2 line provides a valuable model for studying immunological responses against allogeneic T cells and serves as a prototype for developing specific cell types for future cell-based therapy.

Capped F10, a novel DNA directed fluoropyrimidine, in TP53 mutated acute myeloid leukemia (AML).

7046 Background: Acute myeloid leukemia (AML) is an aggressive malignancy characterized by resistance to treatment and poor outcomes. One of the most devastating mutations in AML involves loss of the tumor suppressor TP53. AML harboring this loss carries a dismal prognosis and there is a clear need for new approaches. Methods: To identify new therapeutics, a TP53 null and WT isogenic cell line pair was generated using CRISPR/CAS9 using a murine AML cell line. Clonal isolates were obtained and loss of detectable TP53 was confirmed by Western Blotting. A high throughput screen of currently FDA approved anticancer agents was performed. Results: When agents were ranked by equal cytotoxicity against TP53 null and WT AML cells 20% (6/30) of the top 30 agents targeted the folate/thymidylate synthase axis. This suggests that thymidylate synthase (TS) is a vulnerability in TP53 null AML cells. To confirm TS as a target in TP53 null AML an additional murine AML TP53 null/WT cell line pair was generated using CRISPR/CAS9. When these cell lines were treated with doxorubicin the TP53 null cells were significantly more resistant (p≤0.001 at 10nM). Capped F10 (CF10) is a second generation fluoropyrimidine oligonucleotide that has been shown to be a highly potent inhibitor of TS. In contrast to doxorubicin, CF10 treatment resulted in significant cell death when dosed in the low picomolar range with minimal differences between the null and WT cell lines (p = 0.31 at 50pM). To extend this result TP53 WT or null cells were injected into syngeneic recipients and upon engraftment mice were treated with CF10 at 300mg/kg twice weekly. CF10 provided a significant survival benefit in the TP53 null but not WT injected mice consistent with CF10 having activity against TP53 null AML (p = 0.035 vs 0.13). To confirm a TP53 independent mechanism, competition assays were performed with CAS9 expressing AML cells partially infected with a vector expressing an sgRNA targeting TP53 and a GFP reporter. When the mixed population was treated with the IC90 concentration of 5-fluorouracil there was a highly significant increase in GFP+ cells indicating a TP53 mediated cell death (p = < 0.001). In contrast when cells were treated with the IC90 concentration of CF10 there was a non-significant increase in GFP+ population (p = 0.074) consistent with a mainly TP53 independent mechanism. Fluoropyrimidine based therapy can be myelosuppressive especially in patients with low dihydropyrimidine dehydrogenase (DPD) activity. To assess the tolerability of CF10, healthy WAG/RAJ rats were treated with the DPD inhibitor ethynyl uracil at 2mg/kg alone or in combination with either 141 mg/kg CF10 or the molar equivalent of 50 mg/kg 5-flurouracil (5-FU) and weight determined. In contrast to 5-FU, CF10 treated animals did not have a significant change in weight compared to controls. Conclusions: Taken together these data suggest that CF10 is a promising new agent for TP53 null AML.

Combined inhibition of histone deacetylase and cytidine deaminase improves epigenetic potency of decitabine in colorectal adenocarcinomas

BackgroundTargeting the epigenome of cancerous diseases represents an innovative approach, and the DNA methylation inhibitor decitabine is recommended for the treatment of hematological malignancies. Although epigenetic alterations are also common to solid tumors, the therapeutic efficacy of decitabine in colorectal adenocarcinomas (COAD) is unfavorable. Current research focuses on an identification of combination therapies either with chemotherapeutics or checkpoint inhibitors in modulating the tumor microenvironment. Here we report a series of molecular investigations to evaluate potency of decitabine, the histone deacetylase inhibitor PBA and the cytidine deaminase (CDA) inhibitor tetrahydrouridine (THU) in patient derived functional and p53 null colon cancer cell lines (CCCL). We focused on the inhibition of cell proliferation, the recovery of tumor suppressors and programmed cell death, and established clinical relevance by evaluating drug responsive genes among 270 COAD patients. Furthermore, we evaluated treatment responses based on CpG island density.ResultsDecitabine caused marked repression of the DNMT1 protein. Conversely, PBA treatment of CCCL recovered acetylation of histone 3 lysine residues, and this enabled an open chromatin state. Unlike single decitabine treatment, the combined decitabine/PBA treatment caused > 95% inhibition of cell proliferation, prevented cell cycle progression especially in the S and G2-phase and induced programmed cell death. Decitabine and PBA differed in their ability to facilitate re-expression of genes localized on different chromosomes, and the combined decitabine/PBA treatment was most effective in the re-expression of 40 tumor suppressors and 13 genes typically silenced in cancer-associated genomic regions of COAD patients. Furthermore, this treatment repressed expression of 11 survival (anti-apoptotic) genes and augmented expression of X-chromosome inactivated genes, especially the lncRNA Xist to facilitate p53-mediated apoptosis. Pharmacological inhibition of CDA by THU or its gene knockdown prevented decitabine inactivation. Strikingly, PBA treatment recovered the expression of the decitabine drug-uptake transporter SLC15A1, thus enabling high tumor drug-loads. Finally, for 26 drug responsive genes we demonstrated improved survival in COAD patients.ConclusionThe combined decitabine/PBA/THU drug treatment improved drug potency considerably, and given their existing regulatory approval, our findings merit prospective clinical trials for the triple combination in COAD patients.

Characterization, functional validation, and analysis of the cross-reactive T cell repertoire in the context of heterosubtypic influenza A virus infection

Abstract The T cell receptor (TCR) repertoire is extremely diverse and plays an instrumental role in fighting off pathogens individuals encounter in a lifetime. A key aspect of the immune system combatting the diverse array of pathogens an individual sees may be TCR cross-reactivity, where a TCR can recognize more than one peptide displayed on an MHC-I molecule. Specifically, cross-reactive TCRs may play a key role in allowing the immune system to adapt to rapidly evolving pathogens, such as influenza A virus (IAV). Utilizing 6 variant influenza viruses for priming and secondary challenge, our preliminary data has shown that cross-reactive TCRs seen in primary infection with one IAV can mount a robust and effective memory response to a different IAV, across a range of mutations. However, selection for cross-reactive receptors over multiple infections may create a narrowed repertoire consisting of a few clonotypes, covering a narrow range of antigenic space representing the mutant epitopes encountered. Our data shows that cross-reactive TCRs are more similar to each other than single epitope specific cells and that cross-reactivity can be replicated during peptide stimulation when cross-reactive TCRs are transduced into TCR null cell lines invitro. Given the narrowing nature of cross-reactive responses elicited over different infections, selection for cross-reactivity may provide a substantial escape opportunity with future antigenic variations. Ongoing work focuses on how repeated IAV challenges shape the repertoire, and how previous infections inform future responses by selected cross-reactive T cell pools. Supported by NIH Grant R01112404040 and the St. Jude Graduate School of Biomedical Sciences

Abstract 6431: Targeting Rab7-mediated STING degradation to amplify therapeutic STING agonism in TNBC

Abstract STING (Stimulator of interferon genes), encoded by TMEM173, is a critical regulator of the innate immune response to cytoplasmic double stranded DNA in cancer and is actively being pursued as a target for cancer immunotherapy. STING agonist analogues of 2’-3’cGAMP, the cyclic dinucleotide that directly activates STING downstream of cyclic-GAMP synthase, have entered phase 1 clinical trials for triple negative breast cancers (TNBC). Recently, we have identified tumor cell Phosphatase and tensin homolog (PTEN) inactivation as a precision-based context in which to apply STING agonists. Mechanistically, PTEN null TNBCs maintain Ras-related protein Rab-7a (Rab7) in an inactive state and therefore trafficking of STING to the lysosome is impaired and cells are rendered hypersensitive to STING agonism. Tissue microarray analysis reveals high STING expression in PTEN null TNBCs. Consistently, PTEN null TNBC cell lines were hyper-responsive to STING agonists with increased downstream production of IRF3 targets including CXCL10. In contrast, PTEN wild-type cells exhibit low STING expression and modest sensitivity to STING agonism. As such, although a significant proportion of TNBCs are PTEN null, we sought to broaden these findings to PTEN wild-type TNBCs by genetically and pharmacologically mimicking the PTEN null state by Rab7 knockout and use of a novel selective Rab7 inhibitor, CID1067700. Indeed, using the PTEN wild-type TNBC cell line, MDA-MB-231, we found that PTEN deletion robustly increased STING protein expression. Rab7 CRISPR-Cas9 knockout or treatment of MDA-MB-231 cells with CID1067700 and the STING agonist (ADU-S100) impaired trafficking of STING for lysosomal degradation, upregulated T cell chemokines, and enhanced growth inhibition. Next, to model the potential in vivo implications for immune cell recruitment and function, we used a 3D microfluidic T-cell migration assay. We found that the enhanced CXCL10 levels resulting from addition of CID1067700 to MDA-MB-231 cells led to T-cell recruitment to the tumor spheroids. Taken together, our study identifies PTEN null status as a specific genomic context in TNBC that has exquisite sensitivity to STING agonist therapy and combination with Rab7 inhibition could amplify therapeutic STING agonism in PTEN wild-type TNBCs. Citation Format: Yuqing Zhang, Jessica L. Ritter, Tran C. Thai, Deborah A. Dillon, David A. Barbie, Thanh U. Barbie. Targeting Rab7-mediated STING degradation to amplify therapeutic STING agonism in TNBC. [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 6431.

MP04-12 PHARMACOLOGICAL INHIBITION OF THE MOLECULAR CHAPERONE HSP70 OVERCOMES BELZUTIFAN RESISTANCE IN CCRCC

You have accessJournal of UrologyCME1 Apr 2023MP04-12 PHARMACOLOGICAL INHIBITION OF THE MOLECULAR CHAPERONE HSP70 OVERCOMES BELZUTIFAN RESISTANCE IN CCRCC Michael Basin, Sarah Backe, Michael Bratslavsky, Mark Woodford, Dimitra Bourboulia, Mehdi Mollapour, and Gennady Bratslavsky Michael BasinMichael Basin More articles by this author , Sarah BackeSarah Backe More articles by this author , Michael BratslavskyMichael Bratslavsky More articles by this author , Mark WoodfordMark Woodford More articles by this author , Dimitra BourbouliaDimitra Bourboulia More articles by this author , Mehdi MollapourMehdi Mollapour More articles by this author , and Gennady BratslavskyGennady Bratslavsky More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patients with von-Hippel Lindau (VHL) mutations are predisposed to developing clear cell renal cell carcinoma (ccRCC) through constitutive activation of transcription factor hypoxia-inducible factor 2a (HIF2a). The HIF2a antagonist Belzutifan has recently been FDA approved for treatment VHL disease associated tumors. Results of the recent clinical trial (MK-4682) revealed that a certain proportion of VHL patients progress on therapy. Our objective here was to decipher the molecular mechanisms of Belzutifan mediated downregulation of HIF2a and therefore provide better understanding of drug resistance. The molecular chaperone Hsp70 promotes the stability of hundreds of client proteins, thereby protecting them from degradation. Since HIF2a interacts with Hsp70, we hypothesized that Hsp70 maintains HIF2a stability in ccRCC and is a potential alternative therapeutic target in cases of Belzutifan resistance. METHODS: Cell line models of VHL-null ccRCC (786-0, A498, 769-P) were treated with 2 mM Belzutifan. HIF2a expression levels were evaluated by immunoblotting. Wild-type (WT) or Belzutifan resistant (S304M, G323E) HIF2a were transiently expressed in 786-O cells prior to treatment with Belzutifan or an Hsp70 inhibitor (JG-98). HIF2a degradation was examined by immunoblot. RESULTS: Here, we show that HIF2a is an Hsp70 client in VHL null ccRCC cell line 786-O. Further, we demonstrated a time dependent decrease in HIF2a expression with Belzutifan (2 mM) as early as 2 hours after treatment in three VHL null ccRCC cell lines 786-0, A498, and 769-P cells. Pretreatment of ccRCC cells with the proteasome inhibitor bortezomib blocked Belzutifan mediated downregulation of HIF2a, indicating Belzutifan leads to proteasomal degradation of HIF2a through a VHL independent pathway. Utilizing previously characterized Belzutifan resistant HIF2a-S304M and -G323E mutants we demonstrated that treating ccRCC cells with Hsp70 inhibitor JG-98 leads to mutant-HIF2a degradation, therefore overcoming Belzutifan resistance. CONCLUSIONS: The FDA-approved HIF2a inhibitor Belzutifan causes proteasomal degradation of WT-HIF2a, but not of resistant mutants. Our data indicates that HIF2a stability is reliant on the molecular chaperone Hsp70. Further, treatment with an Hsp70 inhibitor could potentially be used in the clinic to overcome resistance to Belzutifan in patients with VHL disease and ccRCC. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e37 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michael Basin More articles by this author Sarah Backe More articles by this author Michael Bratslavsky More articles by this author Mark Woodford More articles by this author Dimitra Bourboulia More articles by this author Mehdi Mollapour More articles by this author Gennady Bratslavsky More articles by this author Expand All Advertisement PDF downloadLoading ...

Knockout Mutants of OsPUB7 Generated Using CRISPR/Cas9 Revealed Abiotic Stress Tolerance in Rice.

Plants produce and accumulate stress-resistant substances when exposed to abiotic stress, which involves a protein conversion mechanism that breaks down stress-damaged proteins and supplies usable amino acids. Eukaryotic protein turnover is mostly driven by the ubiquitination pathway. Among the three enzymes required for protein degradation, E3 ubiquitin ligase plays a pivotal role in most cells, as it determines the specificity of ubiquitination and selects target proteins for degradation. In this study, to investigate the function of OsPUB7 (Plant U-box gene in Oryza sativa), we constructed a CRISPR/Cas9 vector, generated OsPUB7 gene-edited individuals, and evaluated resistance to abiotic stress using gene-edited lines. A stress-tolerant phenotype was observed as a result of drought and salinity stress treatment in the T2OsPUB7 gene-edited null lines (PUB7-GE) lacking the T-DNA. In addition, although PUB7-GE did not show any significant change in mRNA expression analysis, it showed lower ion leakage and higher proline content than the wild type (WT). Protein-protein interaction analysis revealed that the expression of the genes (OsPUB23, OsPUB24, OsPUB66, and OsPUB67) known to be involved in stress increased in PUB7-GE and this, by forming a 1-node network with OsPUB66 and OsPUB7, acted as a negative regulator of drought and salinity stress. This result provides evidence that OsPUB7 will be a useful target for both breeding and future research on drought tolerance/abiotic stress in rice.

Abstract P2-26-13: Gene deletion of insulin receptor in MCF-7L breast cancer cells by CRISPR gene editing

Abstract The type I insulin-like Growth Factor Receptor (IGF-IR) plays important roles in breast cancer cell proliferation and metastasis. However, preclinical and clinical research targeting IGF-IR also revealed the potential for insulin receptor (IR) to compensate for IGF-1R inhibition. IR is expressed as two isoforms (IRA and IRB) through alternative splicing. The IRB is expressed in adult tissues and thought responsible for cell metabolism while IRA is expressed in fetal development to enhance cell growth. Since breast cancer cells usually express both IGF-IR and IRA/IRB, differential signaling by IRA and IRB has not been clearly demonstrated. To further define the role for IR, we used CRISPR technology for gene editing in exon 2 of IR in MCF-7L cells to delete IR. Cells were selected for absent IR expression by flow cytometry and Sanger sequencing confirmed that one of the clones (CL35) had the targeted 4 base pair deletions in exon 2. Immunoblotting using an antibody to the  subunit of IR showed no expression in CL35. Unexpectedly, 10 nM insulin exposure resulted in downstream AKT and Erk1,2 phosphorylation. Immunoprecipitation showed that IGF-IR was activated by the insulin in CL35 cells. Insulin signaling in CL35 through IGF-IR was inhibited by huEM164, an anti IGF-IR monoclonal antibody. Compared to MCF-7L parental cells, CL35 was more sensitive to IGF-I and less sensitive to insulin as demonstrated by downstream AKT phosphorylation and MTT monolayer growth assay. Insulin stimulated monolayer growth of CL35 measured by MTT was not affected in testing serum free media (SFM) unless transferrin was deleted from the media. These data suggest IGF-IR can function to activate signaling in response to insulin. To ensure that our clone does not contain alternate splicing products resulting in aberrant IR proteins, we are further sequencing IR mRNA in CL35. In conclusion, we have established an IR null breast cancer cell line with reduced sensitivity to insulin and increased sensitivity to IGF-I. This cell line can be used to express the IRA and IRB isoforms to characterize their function in breast cancer cells. Citation Format: Xihong Zhang, Marvis Monteiro, Douglas Yee. Gene deletion of insulin receptor in MCF-7L breast cancer cells by CRISPR gene editing [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-13.

Toward reducing the immunogenic potential of wheat flour: identification and characterization of wheat lines missing omega-5 gliadins encoded by the 1D chromosome.

Eleven wheat lines that are missing genes for the 1D-encoded omega-5 gliadins will facilitate breeding efforts to reduce the immunogenic potential of wheat flour for patients susceptible to wheat allergy. Efforts to reduce the levels of allergens in wheat flour that cause wheat-dependent exercise-induced anaphylaxis are complicated by the presence of genes encoding omega-5 gliadins on both chromosomes 1B and 1D of hexaploid wheat. In this study, we screened 665 wheat germplasm samples using gene specific DNA markers for omega-5 gliadins encoded by the genes on 1D chromosome that were obtained from the reference wheat Chinese Spring. Eleven wheat lines missing the PCR product corresponding to 1D omega-5 gliadin gene sequences were identified. Two of the lines contained the 1BL·1RS translocation. Relative quantification of gene copy numbers by qPCR revealed that copy numbers of 1D omega-5 gliadins in the other nine lines were comparable to those in 1D null lines of Chinese Spring, while copy numbers of 1B omega-5 gliadins were like those of Chinese Spring. 2-D immunoblot analysis of total flour proteins from the selected lines using a specific monoclonal antibody against the N-terminal sequence of omega-5 gliadin showed no reactivity in regions of the blots containing previously identified 1D omega-5 gliadins. Interestingly, RP-UPLC analysis of the gliadin fractions of the selected lines indicated that the expression of omega-1,2 gliadins was also significantly reduced in seven of the lines, implying that 1D omega-5 gliadin and 1D omega-1,2 gliadin genes are tightly linked on the Gli-D1 loci of chromosome 1D. Wheat lines missing the omega-5 gliadins encoded by the genes on 1D chromosome should be useful in future breeding efforts to reduce the immunogenic potential of wheat flour.

Imaginal disk growth factors are Drosophila chitinase-like proteins with roles in morphogenesis and CO2 response.

Chitinase-like proteins (CLPs) are members of the family 18 glycosyl hydrolases, which include chitinases and the enzymatically inactive CLPs. A mutation in the enzyme's catalytic site, conserved in vertebrates and invertebrates, allowed CLPs to evolve independently with functions that do not require chitinase activity. CLPs normally function during inflammatory responses, wound healing, and host defense, but when they persist at excessive levels at sites of chronic inflammation and in tissue-remodeling disorders, they correlate positively with disease progression and poor prognosis. Little is known, however, about their physiological function. Drosophila melanogaster has 6 CLPs, termed Imaginal disk growth factors (Idgfs), encoded by Idgf1, Idgf2, Idgf3, Idgf4, Idgf5, and Idgf6. In this study, we developed tools to facilitate characterization of the physiological roles of the Idgfs by deleting each of the Idgf genes using the CRISPR/Cas9 system and assessing loss-of-function phenotypes. Using null lines, we showed that loss of function for all 6 Idgf proteins significantly lowers viability and fertility. We also showed that Idgfs play roles in epithelial morphogenesis, maintaining proper epithelial architecture and cell shape, regulating E-cadherin and cortical actin, and remarkably, protecting these tissues against CO2 exposure. Defining the normal molecular mechanisms of CLPs is a key to understanding how deviations tip the balance from a physiological to a pathological state.

Transcriptome and Metabolite Profiling of Tomato SGR-Knockout Null Lines Using the CRISPR/Cas9 System.

Stay-green 1 (SGR1) protein is a critical regulator of chlorophyll degradation and senescence in plant leaves; however, the functions of tomato SGR1 remain ambiguous. Here, we generated an SGR1-knockout (KO) null line via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-mediated gene editing and conducted RNA sequencing and gas chromatography−tandem mass spectrometry analysis to identify the differentially expressed genes (DEGs). Solanum lycopersicum SGR1 (SlSGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid levels than those in the wild-type (WT) fruit. Differential gene expression analysis revealed 728 DEGs between WT and sgr#1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, with fold-change >2 and adjusted p-value < 0.05. Most of the DEGs have functions related to photosynthesis, chloroplasts, and carotenoid biosynthesis. The strong changes in pigment and carotenoid content resulted in the accumulation of key primary metabolites, such as sucrose and its derivatives (fructose, galactinol, and raffinose), glycolytic intermediates (glucose, glucose-6-phosphate, and fructose-6-phosphate), and tricarboxylic acid cycle intermediates (malate and fumarate) in the leaves and fruit of the SGR-KO null lines. Overall, the SGR1-KO null lines developed here provide new evidence for the mechanisms underlying the roles of SGR1 as well as the molecular pathways involved in photosynthesis, chloroplasts, and carotenoid biosynthesis.

Lorentz-invariant topological structures of the electromagnetic field in a Fabry-Perot resonant slit grating

The authors construct an absolute (Lorentz-invariant) topology of the electromagnetic field that transcends the boundary between space and time. For a resonant slit grating, they observe null straight lines in the plane wave region, rounded rhombuses in the interference region inside the grating, and a fractal pattern in the evanescent field region where the light funneling occurs.

27 Entinostat restores responsiveness to olaparib in ID8 TP53 null and ID8 TP53 null/BRCA2 null mouse ovarian cancer cell lines

Poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) reduce disease progression in patients diagnosed with ovarian cancer. The anticancer effects of PARPi are most effective in tumors with BRCA mutations and homologous recombination (HR) deficiency (HRD). One mechanism of PARPi resistance is through restoration of HR repair. Our group has shown that entinostat (an HDACi) potentiates the effects of olaparib (a PARPi). The objective of this study was to investigate mechanisms of intrinsic PARPi resistance by studying the combination of entinostat and olaparib in HR proficient and HR deficient mouse ovarian cancer cell lines. We used the ID8 TP53 null (HR proficient) and ID8 TP53 null/BRCA2 null (HR deficient) mouse ovarian cancer cell lines that harbor molecular features consistent with human high-grade serous ovarian cancer. To recapitulate an ongoing clinical trial using this drug combination, cells were pre-treated with 0.25 μM Entinostat or control for 24 hours, followed by 24 or 72 hours of drug treatment [0.5 μM Entinostat, 10 μM of Olaparib, the combination of both, or control]. Clonogenicity assays were used to assess colony formation and MTS assays were used to measure cell viability and proliferation. We then performed immunofluorescence (IF) staining for RAD51 (a marker for HR repair), γH2AX (a marker of DNA damage) and Ki67 (a marker of proliferation). As expected in the clonogenicity assay, there was no statistically significant difference in cell survival between the olaparib treated ID8 TP53 null (HR proficient) cells and vehicletreated controls (p=0.0993; paired t test). Alternatively, the ID8 TP53 null/BRCA2 null (HR deficient) cell line treated with olaparib alone reduced cell viability compared to control (p=0.0245; paired t test). Clonogenicity assays also demonstrated that the combination of entinostat and olaparib reduced colony formation compared to control in the ID8 TP53 null cell line (p<0.0001; one-way ANOVA). MTS assays corroborated this treatment effect (p<0.0001; two way ANOVA). Preliminary data suggests that compared to controls and each drug alone, IF staining for RAD51 and Ki67 expression was reduced in cells treated with combination therapy. Also preliminarily, there was increased number of γH2AX foci in combination treatment compared to controls and each drug alone. Entinostat restores responsiveness to olaparib in ID8 TP53 null HR proficient mouse ovarian cancer cells that molecularly resemble high-grade serous ovarian carcinoma. These results suggest this drug combination can overcome intrinsic resistance to PARPi and provide additional preclinical support for clinical investigation of this combination for the treatment of HR proficient ovarian cancer.

SORL1 regulates the generation of APOE selectively in human neurons

AbstractBackgroundSORL1 is a neuronal sorting receptor that has been strongly implicated in the pathogenesis of Alzheimer’s disease (AD). While SORL1 has structural similarities to low‐density lipoprotein (LDL) receptors, it is unclear if it plays a role in regulating lipoprotein levels in the brain. We aimed to utilize human induced pluripotent stem cells (iPSCs) to understand the intersection of SORL1 and APOE biology and interrogate the potential role of SORL1 as an LDL receptor.MethodWe differentiated iPSC lines derived from the ROS and MAP cohorts that span the cognitive and neuropathological spectrum of aging to neuron and astrocyte fates and performed unbiased proteomic profiling, Western blotting, and ELISA to quantify protein levels of SORL1 and APOE. We next used CRISPR/Cas9 to generate SORL1 null iPSC lines and differentiated to neuron, astrocyte, and microglial fates. In neurons and astrocytes, AD‐relevant phenotypes such as APOE, Abeta, and phospho‐tau were assessed. Rescue experiments were performed in SORL1 KO neurons using pharmacological perturbations and shRNA to elucidate the pathways downstream of loss of SORL1 that lead to altered Abeta, tau and APOE.ResultWe observed a strong correlation between SORL1 and APOE protein expression in neurons derived from ROSMAP cohort individuals that was not apparent in astrocytes. Next, we validated previously reported findings that loss of SORL1 in neurons results in an elevation in Abeta levels. We expanded upon these findings to show that SORL1 null neurons display elevated phospho‐tau levels and a significant reduction of extracellular and intracellular APOE. While enhancing retromer and autophagy function rescued the elevation of phosphorylated tau in SORL1 null neurons, these treatments did not rescue reduced APOE levels. Rather, loss of SORL1 resulted in a reduction in APOE RNA expression, suggesting that loss of SORL1 leads to a downregulation of APOE at the transcriptional level. Integration of profiling data from ROSMAP iNs and SORL1 knock out iNs implicate the transcription factors NFkB and REV‐ERB as potentially mediating the effect of loss of SORL1 on APOE RNA levels.ConclusionSORL1 has a neuron‐specific function in regulating APOE levels that is separable from its role in regulating Abeta and phospho‐tau levels.

Abstract C065: Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer

Abstract Background: Unfortunately, the impact of immunotherapy in pancreatic cancer has, to date, been limited. Nonetheless, despite lack of success with checkpoint blockade alone, there is hope that combinations of agents may unlock the potential for immunotherapy success in pancreatic cancer. FAK (Focal Adhesion Kinase) is a non-receptor tyrosine kinase that is elevated in human Pancreatic Ductal Adenocarcinoma (PDAC) tissue and correlates with high levels of fibrosis and poor CD8 cytotoxic T cell infiltration. We, and others, have already demonstrated a role for FAK in promoting tumor evasion by inducing an immunosuppressive microenvironment, specifically by regulation of cytokines such as CCL5. This has led to trials of the FAK inhibitor (defactinib) in conjunction with immunotherapy. Here we set out to improve our understanding of the role of FAK in regulating chemokine/cytokine mediated signaling in PDAC with a view to identify clinically targetable pathways with potential therapeutic benefit to pancreatic cancer patients. Methods: We combined CRISPR, NanoString, Forward Phase Protein Arrays (FPPA) and ELISA with in vivo experiments using a transplantable KrasG12Dp53R172H mouse model of Pancreatic Cancer and validated findings using human patient-derived PDAC cell lines. Results: We used CRISPR-Cas9 gene editing to deplete FAK gene expression in Panc47 cells, a syngeneic cell line isolated from PDAC arising in fully back-crossed C57BL/6 KPC mice, and reconstituted wild-type FAK (FAK-WT) expression into a clonal Panc47 FAK null (FAK-/-) cell line. Subsequent NanoString and FPPA analyses of secreted cytokines in Panc47 FAK-WT and Panc47 FAK-/- cell lines identified CXCL16 as one of the most significantly increased chemokines upon FAK depletion. Similar results were observed when CRISPR mediated FAK depletion was performed in patient-derived PDAC cell lines. Previous work in our lab has shown that depletion of Signal Transducer and Activator of Transcription-3 (STAT3), a key mediator of immune evasion, in combination with FAK depletion in orthotopically implanted PDAC tumors significantly increases CD8 T-cell infiltration into murine tumors when compared with FAK-/- tumors alone. Here we identify that dual FAK/STAT3 depletion further elevates CXCL16 expression and show that CXCR6, the receptor for CXCL16, is associated with both murine and human CD8 T-cells. Conclusions: These findings identify a FAK-dependent CXCL16:CXCR6 paracrine signaling axis associated with elevated recruitment of CD8 T-cells into PDAC tumors. This requires the dual targeting of FAK and STAT3, identifying a novel therapeutic strategy that warrants further investigation. Citation Format: Jane L. Parry, Marta Canel, Alan Serrels. Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C065.

Detailed characterizations of cranial nerve anatomy in E14.5 mouse embryos/fetuses and their use as reference for diagnosing subtle, but potentially lethal malformations in mutants.

Careful phenotype analysis of genetically altered mouse embryos/fetuses is vital for deciphering the function of pre- and perinatally lethal genes. Usually this involves comparing the anatomy of mutants with that of wild types of identical developmental stages. Detailed three dimensional information on regular cranial nerve (CN) anatomy of prenatal mice is very scarce. We therefore set out to provide such information to be used as reference data and selected mutants to demonstrate its potential for diagnosing CN abnormalities. Digital volume data of 152 wild type mice, harvested on embryonic day (E)14.5 and of 18 mutants of the Col4a2, Arid1b, Rpgrip1l and Cc2d2a null lines were examined. The volume data had been created with High Resolution Episcopic Microscopy (HREM) as part of the deciphering the mechanisms of developmental disorders (DMDD) program. Employing volume and surface models, oblique slicing and digital measuring tools, we provide highly detailed anatomic descriptions of the CNs and measurements of the diameter of selected segments. Specifics of the developmental stages of E14.5 mice and anatomic norm variations were acknowledged. Using the provided data as reference enabled us to objectively diagnose CN abnormalities, such as abnormal formation of CN3 (Col4a2), neuroma of the motor portion of CN5 (Arid1b), thinning of CN7 (Rpgrip1l) and abnormal topology of CN12 (Cc2d2a). Although, in a first glimpse perceived as unspectacular, defects of the motor CN5 or CN7, like enlargement or thinning can cause death of newborns, by hindering feeding. Furthermore, abnormal topology of CN12 was recently identified as a highly reliable marker for low penetrating, but potentially lethal defects of the central nervous system.