Knock-in Cell Lines Research Articles

Abstract A084: CDK12 inhibition drives mitotic catastrophe, triggering genome instability and innate immune response in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited therapies. In our screening using patient-derived organoids, we identified cyclin-dependent kinase 12 (CDK12) as a promising target in a subset of PDAC. Both pharmacological and genetic inhibition of CDK12 exerted potent antitumor activity in vitro and in vivo, inducing double-strand DNA breaks and apoptosis. CDK12 is a member of cyclin-dependent kinases that is involved in gene expression regulations at multiple levels. Recently, CDK12 has gained attention as a therapeutic target in several types of cancers including triple-negative breast cancer and Ewing sarcoma. However, little is known about the impact and possibility of CDK12 inhibition in pancreatic cancer treatment. As the lack of specific inhibitors that selectively target CDK12, studying CDK12 has long been challenging. To overcome this, we used CRISPR-Cas9 mediated genome editing to generate FKBP12F36V (dTAG) knock-in PDAC cell lines, enabling rapid elimination of endogenous CDK12 using a degrader against dTAG (dTAG-13). Quantitative-image-based cytometry and time lapse imaging showed that CDK12 ablation induces mitotic arrest, DNA damage and apoptosis (mitotic catastrophe). Specifically, loss of CDK12 kinase activity in the S phase was sufficient to slow DNA replication speed, leading to spindle assembly checkpoint activation and mitotic catastrophe. Importantly, cells that escaped mitotic death accumulate micronuclei, triggering immune response signaling that is mediated by the cGAS-STING-type I interferon pathway. Our results suggest that CDK12 is a promising target in a subset of PDAC patients, where CDK12 inhibition exerts antitumor activity both in cell autonomous and non-cell autonomous ways through inducing mitotic catastrophe as well as triggering immune response. Citation Format: Dosuke Iwadate, Keisuke Yamamoto, Hiroyuki Kato, Keisuke Tateishi, Mitsuhiro Fujishiro. CDK12 inhibition drives mitotic catastrophe, triggering genome instability and innate immune response in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A084.

Initial Characterization of WDR5B Reveals a Role in the Proliferation of Retinal Pigment Epithelial Cells.

The chromatin-associated protein WDR5 has been widely studied due to its role in histone modification and its potential as a pharmacological target for the treatment of cancer. In humans, the protein with highest sequence homology to WDR5 is encoded by the retrogene WDR5B, which remains unexplored. Here, we used CRISPR-Cas9 genome editing to generate WDR5B knockout and WDR5B-FLAG knock-in cell lines for further characterization. In contrast to WDR5, WDR5B exhibits low expression in pluripotent cells and is upregulated upon neural differentiation. Loss or shRNA depletion of WDR5B impairs cell growth and increases the fraction of non-viable cells in proliferating retinal pigment epithelial (RPE) cultures. CUT&RUN chromatin profiling in RPE and neural progenitors indicates minimal WDR5B enrichment at established WDR5 binding sites. These results suggest that WDR5 and WDR5B exhibit several divergent biological properties despite sharing a high degree of sequence homology.

VCP activator reverses nuclear proteostasis defects and enhances TDP-43 aggregate clearance in multisystem proteinopathy models.

Pathogenic variants in valosin-containing protein (VCP) cause multisystem proteinopathy (MSP), a disease characterized by multiple clinical phenotypes including inclusion body myopathy, Paget's disease of the bone, and frontotemporal dementia (FTD). How such diverse phenotypes are driven by pathogenic VCP variants is not known. We found that these diseases exhibit a common pathologic feature: ubiquitinated intranuclear inclusions affecting myocytes, osteoclasts, and neurons. Moreover, knock-in cell lines harboring MSP variants show a reduction in nuclear VCP. Given that MSP is associated with neuronal intranuclear inclusions comprised of TDP-43 protein, we developed a cellular model whereby proteostatic stress results in the formation of insoluble intranuclear TDP-43 aggregates. Consistent with a loss of nuclear VCP function, cells harboring MSP variants or cells treated with VCP inhibitor exhibited decreased clearance of insoluble intranuclear TDP-43 aggregates. Moreover, we identified 4 compounds that activate VCP primarily by increasing D2 ATPase activity, where pharmacologic VCP activation appears to enhance clearance of insoluble intranuclear TDP-43 aggregate. Our findings suggest that VCP function is important for nuclear protein homeostasis, that impaired nuclear proteostasis may contribute to MSP, and that VCP activation may be a potential therapeutic by virtue of enhancing the clearance of intranuclear protein aggregates.

Abstract PO4-14-12: Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids

Abstract Background: Activating mutations in ERBB2 (HER2) are enriched >4-fold in invasive lobular carcinoma (ILC) with a rate of up to 19% in metastatic ILC. ILC is a histologic subtype of breast cancer characterized by loss of E-cadherin (CDH1), suggesting a potential interaction between loss of CDH1 and mutations in ERBB2. Recent trials have demonstrated promising single agent efficacy using the irreversible pan-HER tyrosine kinase inhibitor (TKI), neratinib, in patients with metastatic ERBB2 mutant ILC. However, further studies on combination therapies with other anticancer agents are needed to increase response rate and progression free survival for these patients. HER2-targeted antibody drug conjugates (ADC), particularly trastuzumab deruxtecan (T-DXd), have shown great promise in HER2-low metastatic breast cancer. Yet, their efficacy as a single agent or with HER2 TKIs in HER2-low and -mutant ILC is unknown and warrants investigation. Methods: Past studies analyzing ERBB2 mutations used overexpression of ERBB2 mutant cDNA, but this approach does not faithfully recapitulate the human disease. To model ERBB2 missense mutations as found in human breast cancers, we used CRISPR-based prime editing to generate a panel of isogenic ILC cell lines and patient-derived organoids (PDO) harboring ERBB2 wild-type (WT) or ERBB2 mutations (S310F or V777L). Both of these ERBB2 mutations have been previously characterized and are known to be activating mutations. We then used them to test neratinib and other TKIs with ADCs, including T-DXd and trastuzumab emtansine (T-DM1). Results: We successfully introduced single copy, heterozygous activating ERBB2 mutations (S310F or V777L) into two ERBB2-nonamplified metastatic ILC cell lines (MDA-MB-134 and SUM44PE) and one ERBB2-nonamplified metastatic ILC PDO (IPM-BO-053). Positive clones carrying the mutations were verified by Sanger sequencing and droplet digital PCR and subsequently pooled together. We further demonstrated that these mutations hyperactivated HER2 and downstream signaling pathways. ILC cell lines harboring these mutations showed enhanced sensitivity to HER2 TKIs but not ADCs. In contrary, ERBB2 mutations did not alter responses of IPM-BO-053 PDOs to HER2 TKIs but significantly increased responses to ADCs. Interestingly, we also observed accelerated HER2 protein degradation upon heregulin stimulation in ERBB2 mutant ILC PDOs, suggesting activating ERBB2 mutations may enhance ADC/HER2 complex internalization, degradation, and release of payloads. Lastly, we explored drug synergy between HER2 TKIs and ADCs in isogenic IPM-BO-053 PDOs and found that combination of T-DXd and neratinib or afatinib showed synergy (Combination Index < 1). Conclusions: Although the reason for the discrepancies in drug response between ILC cell lines and PDOs is not clear, we hypothesize that response in 3D PDOs might be more faithfully representing response seen in patients. We will generate additional ILC PDOs with knock-in ERBB2 mutations to validate our findings. Irreversible HER2 TKIs, such as neratinib and afatinib, showed synergy with T-DXd in ERBB2 mutant ILC PDOs. This holds important therapeutic implications in light of current treatment options for ILC. In future experiments, we will test if neratinib or afatinib increases endocytic uptake of T-DXd using a fluorescently labeled endocytosis tracker. Further, an in-depth molecular characterization of our isogenic cell lines and PDOs models is ongoing to gain mechanistic insights into how activating ERBB2 mutations increase HER2 internalization and degradation. Our in vitro studies provide a strong foundation for in vivo testing of neratinib or afatinib with T-DXd for ERBB2 mutant ILC. Citation Format: Jie Bin Liu, Danielle Tseng, Jagmohan Hooda, Daniel Brown, Adrian Lee, Steffi Oesterreich. Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-12.

Abstract 5609: A WRN screening cascade to facilitate novel drug discovery

Abstract Human RecQ deconjugating enzyme WRN is involved in DNA replication, DNA repair, recombination, transcription and telomere stabilization[1]. It plays a key role in nucleic acid metabolism as well. WRN defects lead to premature aging, type II diabetes, osteoporosis, atherosclerosis and cancer[2]. Hence it is of great interest of both pharmaceutical and academic field to develop the WRN inhibitors. Here we constructed an integrated experimental cascade, which contains both in vitro and in vivo assays, to conduct the high throughput hit-to-lead compound screen. WRN proteins of different length have been successfully purified and utilized to develop multiple biochemical assays such as unwinding assay and ATPase assay. We have also validated different cellular assays, including proliferation and immunofluorescence, to assess the cytotoxicity and the influence of downstream biomarkers of WRN inhibitors. A WRN knock-out cell line has been generated to better appreciate the inhibition mechanism. In addition, we have generated a WRN-HiBiT knock-in cell line to evaluate WRN degraders or target-compound interactions. Lastly, multiple CDX models utilizing different MSI or MSS cell lines have been validated to help determine the efficacy of WRN inhibitors thus shed light on the drug indications. Together our WRN screening cascade can provide comprehensive compound evaluation across in vitro and in vivo platforms, thus serve as an efficient screening platform for new drug discovery. [1] Kitano K. Structural mechanisms of human RecQ helicases WRN and BLM. Front Genet. 2014 Oct 29;5:366. [2]Hussain M, Krishnamurthy S, Patel J, Kim E, Baptiste BA, Croteau DL, Bohr VA. Skin Abnormalities in Disorders with DNA Repair Defects, Premature Aging, and Mitochondrial Dysfunction. J Invest Dermatol. 2021 Apr;141(4S):968-975. Citation Format: Aicheng Wang, Kejun Mao, Tao Li, Lizhao Guan, Yuhong Chen, Haiting Dai, Xian Wu, Jiabao Lv, Xu Wang, Cong Huang, Tiejun Bing. A WRN screening cascade to facilitate novel drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5609.

Impaired binding affinity of YTHDC1 with METTL3/METTL14 results in R-loop accumulation in myelodysplastic neoplasms with DDX41 mutation

DEAD box helicase 41 (DDX41) mutations are the most prevalent predisposition to familial myelodysplastic syndrome (MDS). However, the precise roles of these variants in the pathogenesis of MDS have yet to be elucidated. Here, we discovered a novel mechanism by which DDX41 contributes to R-loop-induced DNA damage responses (DDR) in cooperation with the m6A-METTL complex (MAC) and YTHDC1 using DDX41 knockout (KO) and DDX41 knock-in (KI, R525H, Y259C) cell lines as well as primary samples from MDS patients. Compared to wild type (WT), DDX41 KO and KI led to increased levels of m6A RNA methylated R-loop. Interestingly, we found that DDX41 regulates m6A/R-loop levels by interacting with MAC components. Further, DDX41 promoted the recruitment of YTHDC1 to R-loops by promoting the binding between METTL3 and YTHDC1, which was dysregulated in DDX41-deficient cells, contributing to genomic instability. Collectively, we demonstrated that DDX41 plays a key role in the physiological control of R-loops in cooperation with MAC and YTHDC1. These findings provide novel insights into how defects in DDX41 influence MDS pathogenesis and suggest potential therapeutic targets for the treatment of MDS.

IntAct: A nondisruptive internal tagging strategy to study the organization and function of actin isoforms.

Mammals have 6 highly conserved actin isoforms with nonredundant biological functions. The molecular basis of isoform specificity, however, remains elusive due to a lack of tools. Here, we describe the development of IntAct, an internal tagging strategy to study actin isoforms in fixed and living cells. We identified a residue pair in β-actin that permits tag integration and used knock-in cell lines to demonstrate that IntAct β-actin expression and filament incorporation is indistinguishable from wild type. Furthermore, IntAct β-actin remains associated with common actin-binding proteins (ABPs) and can be targeted in living cells. We demonstrate the usability of IntAct for actin isoform investigations by showing that actin isoform-specific distribution is maintained in human cells. Lastly, we observed a variant-dependent incorporation of tagged actin variants into yeast actin patches, cables, and cytokinetic rings demonstrating cross species applicability. Together, our data indicate that IntAct is a versatile tool to study actin isoform localization, dynamics, and molecular interactions.

High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter

Plant cells serve as versatile platforms for the production of high-value recombinant proteins. This study explored the efficacy of utilizing an endogenous αAmy3 promoter for the expression of a bioactive pharmaceutical protein, specifically the mature region of human bone morphogenetic protein 2 (hBMP2m). Utilizing a refined CRISPR/Cas9-mediated intron-targeting insertion technique, which incorporates an artificial 3’ splicing site upstream of the target gene, we achieved a transformation efficiency of 13.5% in rice calli that carried the rice-codon optimized mature region of hBMP2 cDNA (rhBMP2m) in the αAmy3 intron 1. Both homozygous and heterozygous rhBMP2m knock-in rice suspension cell lines were generated. These lines demonstrated the endogenous αAmy3 promoter regulated rhBMP2m mRNA and rhBMP2m recombinant protein expression, with strongly upregulation in respond to sugar depletion. The homozygous rhBMP2m knock-in cell line yielded an impressive 21.5 μg/mL of rhBMP2m recombinant protein, accounting for 1.03% of the total soluble protein. The high-yield expression was stably maintained across two generations, indicating the genetic stability of rhBMP2m gene knock-in at the αAmy3 intron 1 locus. Additionally, the rice cell-derived rhBMP2m proteins were found to be glycosylated, capable of dimer formation, and bioactive. Our results indicate that the endogenous rice αAmy3 promoter–signal peptide-based expression system is an effective strategy for producing bioactive pharmaceutical proteins.Key points• The endogenous αAmy3 promoter-based expression system enhanced the yield of BMP2• The increased yield of BMP2 accounted for 1.03% of the total rice-soluble proteins• The rice-produced BMP2 showed glycosylation modifications, dimer formation, and bioactivity

Sensitivity to Targeted UBA1 Inhibition in a Myeloid Cell Line Model of Vexas Syndrome

Somatic UBA1 mutations in hematopoietic cells are a hallmark of VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome, which is a late-onset, male-predominant myeloid autoinflammatory disease associated with bone marrow failure and high mortality. UBA1 resides on the X chromosome and encodes the E1 enzyme isoforms UBA1a and UBA1b, which are essential for nuclear and cytosolic protein ubiquitination, respectively. The majority of UBA1 mutations in VEXAS syndrome comprise hemizygous missense mutations affecting methionine-41 (M41), leading to loss of UBA1b, gain of a truncated protein isoform (UBA1c),and globally reduced polyubiquitination. The relationship between impaired polyubiquitination, autoinflammation, and dyspoiesis in VEXAS syndrome is poorly understood, highlighting the need for experimental models that reproduce the genetic, biochemical, morphological, and inflammatory features associated with somatic UBA1 M41 missense mutations. To investigate the effects of VEXAS syndrome-associated UBA1 mutations, we used CRISPR-Cas9-based homology directed repair (HDR) in the male myeloid cell line 32D to generate isogenic single cell clones expressing Uba1 WT or Uba1 M41L from the endogenous mouse Uba1 locus. Sanger sequencing analysis of genomic and complementary DNA indicated the presence of a single Uba1 allele in Uba1 WT and Uba1 M41L single cell clones. Consistent with prior analyses of VEXAS syndrome patient samples, UBA1 and ubiquitin immunoblots showed that hemizygous Uba1 M41L expression was associated with loss of the UBA1b protein isoform, gain of the UBA1c protein isoform and reduced polyubiquitination. Uba1 M41L cells also contained abnormal cytoplasmic vacuoles, which were largely absent from Uba1 WT cells. Electron microscopy revealed that vacuoles in Uba1 M41L cells contained a variety of endolysosomal membranes, including small vesicles, multivesicular bodies, and multilamellar lysosomes. Similar to cytokine profiles in VEXAS syndrome patient samples, concentrations of IL1β and CXCL10 were significantly increased in supernatants from Uba1 M41L cells compared to Uba1 WT cells; Uba1 M41L cell supernatants also contained higher concentrations of CCL5, CCL17, CXCL9, and CXCL12. Thus, hemizygous Uba1 M41L expression in a myeloid cell line recapitulates key biochemical, morphological, and inflammatory features of VEXAS syndrome. The UBA1c protein isoform generated via UBA1 M41 missense mutations has reduced capacity to form the UBA1-ubiquitin thioester bond necessary for ubiquitin transfer to E2 enzymes. We hypothesized that decreased UBA1 activity would render Uba1 M41L cells vulnerable to the UBA1 inhibitor TAK243. In cell proliferation assays, Uba1 M41L cells were more sensitive to TAK243 compared to Uba1 WT cells (IC 50 mean±s.d., 1.4±0.6nM vs. 63±28nM, respectively). TAK243 promoted apoptosis in Uba1 M41L cells, as indicated by increased annexin-V/propidium iodide staining, H2AX, H2AX pS139, and PARP1 cleavage. TAK243 also led to preferential loss of Uba1 M41L cells in competition assays with Uba1 WT cells. To determine if on-target UBA1 inhibition was responsible for the increased TAK243 sensitivity of Uba1 M41L cells, we used CRISPR-HDR to generate a TAK243 drug-binding mutant, Uba1 A580S. Knock-in of Uba1 A580S rendered Uba1 M41L cells resistant to TAK243; the TAK243 IC 50 was significantly higher in Uba1 M41L;A580S cells compared to Uba1 M41L cells (mean±s.d., 73±16nM vs. 3.8±1.2nM, respectively). Together, these data demonstrate that hemizygous expression of Uba1 M41L in 32D cells confers TAK243 sensitivity via on-target inhibition of UBA1. Notably, increased TAK243 sensitivity and reduced polyubiquitination in Uba1 M41L cells were rescued by overexpression of UBA1b, but not UBA1b/C632A (catalytically inactive) or luciferase, indicating that loss of UBA1b enzymatic function contributes to the phenotype conferred by Uba1 M41L. In summary, we present a myeloid cell line with hemizygous expression of Uba1 M41L that exhibits the biochemical, morphological, and inflammatory features of VEXAS syndrome. We find that loss of UBA1b is a cause of reduced polyubiquitination and renders Uba1 M41L cells vulnerable to targeted UBA1 inhibition by TAK243. Our Uba1 M41L knock-in cell line is a faithful model of VEXAS syndrome that will aid in the study of disease pathogenesis and the development of effective therapies.

Human lysyl-tRNA synthetase phosphorylation promotes HIV-1 proviral DNA transcription.

Human lysyl-tRNA synthetase (LysRS) was previously shown to be re-localized from its normal cytoplasmic location in a multi-aminoacyl-tRNA synthetase complex (MSC) to the nucleus of HIV-1 infected cells. Nuclear localization depends on S207 phosphorylation but the nuclear function of pS207-LysRS in the HIV-1 lifecycle is unknown. Here, we show that HIV-1 replication was severely reduced in a S207A-LysRS knock-in cell line generated by CRISPR/Cas9; this effect was rescued by S207D-LysRS. LysRS phosphorylation up-regulated HIV-1 transcription, as did direct transfection of Ap4A, an upstream transcription factor 2 (USF2) activator that is synthesized by pS207-LysRS. Overexpressing an MSC-derived peptide known to stabilize LysRS MSC binding inhibited HIV-1 replication. Transcription of HIV-1 proviral DNA and other USF2 target genes was reduced in peptide-expressing cells. We propose that nuclear pS207-LysRS generates Ap4A, leading to activation of HIV-1 transcription. Our results suggest a new role for nuclear LysRS in facilitating HIV-1 replication and new avenues for antiviral therapy.

Analysis of structure–activity relationship of indol-3-yl-N-phenylcarbamic amides as potent STING inhibitors

A structure–activity relationship (SAR) study of stimulator of interferon gene (STING) inhibition was performed using a series of indol-3-yl-N-phenylcarbamic amides and indol-2-yl-N-phenylcarbamic amides. Among these analogs, compounds 10, 13, 15, 19, and 21 inhibited the phosphorylation of STING and interferon regulatory factor 3 (IRF3) to a greater extent than the reference compound, H-151. All five analogs showed stronger STING inhibition than H-151 on the 2′,3′-cyclic GMP-AMP-induced expression of interferon regulatory factors (IRFs) in a STINGR232 knock-in THP-1 reporter cell line. The half-maximal inhibitory concentration of the most potent compound, 21, was 11.5 nM. The molecular docking analysis of compound 21 and STING combined with the SAR study suggested that the meta- and para-positions of the benzene ring of the phenylcarbamic amide moiety could be structurally modified by introducing halides or alkyl substituents.

Sensitivity to targeted UBA1 inhibition in a myeloid cell line model of VEXAS syndrome

AbstractSomatic UBA1 mutations in hematopoietic cells are a hallmark of Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic (VEXAS) syndrome, which is a late-onset inflammatory disease associated with bone marrow failure and high mortality. The majority of UBA1 mutations in VEXAS syndrome comprise hemizygous mutations affecting methionine-41 (M41), leading to the expression of UBA1M41T, UBA1M41V, or UBA1M41L and globally reduced protein polyubiquitination. Here, we used CRISPR-Cas9 to engineer isogenic 32D mouse myeloid cell lines expressing hemizygous Uba1WT or Uba1M41L from the endogenous locus. Consistent with prior analyses of VEXAS syndrome patient samples, hemizygous Uba1M41L expression was associated with loss of the UBA1b protein isoform, gain of the UBA1c protein isoform, reduced polyubiquitination, abnormal cytoplasmic vacuoles, and increased production of interleukin-1β and inflammatory chemokines. Vacuoles in Uba1M41L cells contained a variety of endolysosomal membranes, including small vesicles, multivesicular bodies, and multilamellar lysosomes. Uba1M41L cells were more sensitive to the UBA1 inhibitor TAK243. TAK243 treatment promoted apoptosis in Uba1M41L cells and led to preferential loss of Uba1M41L cells in competition assays with Uba1WT cells. Knock-in of a TAK243-binding mutation, Uba1A580S, conferred TAK243 resistance. In addition, overexpression of catalytically active UBA1b in Uba1M41L cells restored polyubiquitination and increased TAK243 resistance. Altogether, these data indicate that loss of UBA1b underlies a key biochemical phenotype associated with VEXAS syndrome and renders cells with reduced UBA1 activity vulnerable to targeted UBA1 inhibition. Our Uba1M41L knock-in cell line is a useful model of VEXAS syndrome that will aid in the study of disease pathogenesis and the development of effective therapies.

Live cell transcription-coupled nucleotide excision repair dynamics revisited

Transcription–blocking lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which prevents DNA damage-induced cellular toxicity and maintains proper transcriptional processes. TC-NER is initiated by the stalling of RNA polymerase II (RNAPII), which triggers the assembly of TC-NER-specific proteins, namely CSB, CSA and UVSSA, which collectively control and drive TC-NER progression. Previous research has revealed molecular functions for these proteins, however, exact mechanisms governing the initiation and regulation of TC-NER, particularly at low UV doses have remained elusive, partly due to technical constraints. In this study, we employ knock-in cell lines designed to target the endogenous CSB gene locus with mClover, a GFP variant. Through live cell imaging, we uncover the intricate molecular dynamics of CSB in response to physiologically relevant UV doses. We showed that the DNA damage-induced association of CSB with chromatin is tightly regulated by the CSA-containing ubiquitin-ligase CRL complex (CRL4CSA). Combining the CSB-mClover knock-in cell line with SILAC-based GFP-mediated complex isolation and mass-spectrometry-based proteomics, revealed novel putative CSB interactors as well as discernible variations in complex composition during distinct stages of TC-NER progression. Our work not only provides molecular insight into TC-NER, but also illustrates the versatility of endogenously tagging fluorescent and affinity tags.

417-P: Functional Characterization of a Rare, Pathogenic Missense Variant Implicates FRAS1 as a Novel Gene for Risk of Progression to End-Stage Kidney Disease in Diabetes

Progressive renal decline is the central manifestation of diabetic nephropathy (DN) that ultimately leads to end-stage kidney disease (ESKD). Leveraging an innovative family-based approach that integrates electronic medical record data, a unique population-based genealogy resource, and next-generation sequencing, we recently identified a rare missense variant (p.R2516C, allele frequency=0.00002) in FRAS1, which encodes an extracellular matrix protein that regulates epidermal basement membrane adhesion and mediates glomerular integrity, in a family with diabetes and ESKD. While this rare missense variant is predicted to be deleterious (CADD score=32), whether this impacts FRAS1 function is unknown; here, we set out to assess the functional impact of this variant. To do so, we used CRISPR-Cas9 gene editing technology to generate 3 novel HEK293 cell lines (2 FRAS1 knock-in cell lines (a synonymous p.H2520H control cell line and a missense p.R2516C variant cell line) and a FRAS1 knock-out cell line) and performed in vitro cell migration and cell adhesion assays. Relative to the control cell line, both the FRAS1 knock-out cell line and the missense variant cell line displayed significantly inhibited cell migration (p = 1.54X10-13 and p = 5.05X10-5, respectively). Similarly, cell adhesion was significantly inhibited in cells in which FRAS1 was knocked out and in those carrying the p.R2516C variant compared to the control cell line (p = 6.72X10-9 and p = 6.63X10-6, respectively). Together, these data suggest that the FRAS1 p.R2516C variant negatively impacts FRAS1 function and further implicates this gene as having a potential role in the progression of ESKD in individuals with diabetes. Disclosure T.Chen: None. M.B.Neeley: None. J.M.Lazaro-guevara: None. M.Pezzolesi: None. C.A.Simeone: None. A.Rodan: None. W.L.Holland: Research Support; SymbioCellTech, LLC. M.G.Pezzolesi: Research Support; Renalytix. Funding National Institutes of Health (R01DK128641)

Abstract 3971: Developing cancer cell based in vitro and in vivo models with CRISPR-mediated knock-in technology for anti-tumor drug discovery

Abstract Human cancer cell lines have long been applied for anti-tumor drug discovery considering their acquired genomic diversity of different carcinomas. Genetic engineering based on cancer cell lines is no doubt a useful and promising approach in induction of new therapeutic targets/elements while maintaining intrinsic characteristics of original cell in parallel. Recently, the emergence of CRISPR knock-in technology has accelerated precise editing across whole genome, from gene depletion, small fragment insertions to site-direct mutations, thereby prompting our understanding of the influence of a specific oncotarget in given tumor genetic background. Herein, by employing CRISPR/Cas9 system, two human cancer cell line-based, inheritable drug resistance models were established where the mutation site C481S of BTK in REC-1 conferred resistance to Ibrutinib and the mutation site R537S/D538G of ESR1 exerted resistance to Fulvestrant in MCF-7. In addition, the engineered cell models also enable the mechanism characterization and development of viable therapeutic agents targeting those oncogenic-driver mutations in a subtype of cancers. The murine colon cancer cell line CT26, which harbors KRASG12D mutation, was genetically modified to express KRASG12C protein to allow for an expandable pocket providing opportunities for compound recruitment. The resultant KRASG12C CT26 cell line showed pronounced inhibitory effect after treatment with AMG510. More interestingly, the therapeutic combination involving both AMG510 and anti-PD1 antibodies demonstrated more effective anti-tumor activities when compared to monotherapy. Moreover, CRIPSR-mediated engineered knock-in cells with a traceable tag linked to endogenous oncotarget also exhibit capability in new therapeutic modality discovery. Based on HiBiT protein tagging technology, we generated a series of HiBiT knock-in cell lines for real-time cell-based protein degradation analysis on various compelling targets, such as ESR1, KRAS, and BRD4, et al. Thereby, the construction of these in vitro and in vivo models with the ease of CRISPR KI technology is indispensable in new generation of therapeutic drug exploration. Citation Format: Nengwei Xu, Reifeng Wang, Jian Xiang, Qingyang Gu, Xiangnan Qiang. Developing cancer cell based in vitro and in vivo models with CRISPR-mediated knock-in technology for anti-tumor drug discovery. [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 3971.

Establishment of TUBB3-mCherry knock-in human pluripotent stem cell line using CRISPR/Cas9 (SNUe003-A-4)

TUBB3 is a structural neuronal protein important for multiple neuronal functions including axonal guidance and maturation. This study aimed to generate a human pluripotent stem cell (hPSC) line with a TUBB3-mCherry reporter using CRISPR/SpCas9 nuclease. The stop codon in the last exon of TUBB3 was replaced with a T2A-mCherry cassette using CRISPR/SpCas9-mediated homologous recombination. The established TUBB3-mCherry knock-in cell line exhibited typical pluripotent characteristics. The mCherry reporter faithfully replicated the endogenous level of TUBB3 upon induction of neuronal differentiation. The reporter cell line could contribute to the investigation of neuronal differentiation, neuronal toxicity, and neuronal tracing.

Candidate drugs associated with sensitivity of cancer cell lines with DLST amplification or high mRNA levels

Overexpression of the dihydrolipoamide S-succinyltransferase (DLST) is associated with poor outcome in neuroblastoma patients and triple-negative breast cancer (TNBC) and specifically with the oxidative phosphorylation (OXPHOS) pathway. Inhibitors of OXPHOS were previously suggested as a potential therapeutic strategy for a subset of patients with high-risk neuroblastoma. Here, we tested if cell lines with DLST amplifications or high mRNA levels were associated with sensitivity to 250 drugs from the Genomics of Drug Sensitivity in Cancer (GDSC) dataset by comparing them to cell lines without these changes. DLST-altered cell lines were more sensitive to 7 approved drugs, among these obatoclax mesylate, a BCL2 inhibitor that reduces OXPHOS in human leukemia stem cells. Moreover, several protein kinase inhibitors were identified to be efficient in cell lines with DLST amplifications or high mRNA levels, suggesting a vulnerability of DLST-altered cell lines for drugs targeting the ERK/MAPK pathway. Furthermore, increased DLST expression in cell lines with driver mutations in KRAS supported this relationship. We therefore conclude that, in addition to OXPHOS, protein kinases could be potential targets of therapy in the presence of DLST amplifications or high mRNA levels. The new drug candidates proposed here could serve in experimental testing on drug efficacy in knock-in cell lines and DLST-activated tumors.

Using Optogenetics to Spatially Control Cortical Dynein Activity in Mitotic Human Cells.

Several light-inducible hetero-dimerization tools have been developed to spatiotemporally control subcellular localization and activity of target proteins or their downstream signaling. In contrast to other genetic technologies, such as CRISPR-mediated genome editing, these optogenetic tools can locally control protein localization on the second timescale. In addition, these tools can be used to understand the sufficiency of target proteins' function and manipulate downstream events. In this chapter, I will present methods for locally activating cytoplasmic dynein at the mitotic cell cortex in human cells, with a focus on how to generate knock-in cell lines and set up a microscope system.

Generation of Amber Suppression Cell Lines Using CRISPR-Cas9.

Genetic code expansion via amber suppression allows cotranslational, site-specific introduction of nonnatural chemical groups into proteins in the living cell. The archaeal pyrrolysine-tRNA/pyrrolysine-tRNA synthetase (PylT/RS) pair from Methanosarcina mazei (Mma) has been established for incorporation of a wide range of noncanonical amino acids (ncAAs) in mammalian cells. Once integrated in an engineered protein, ncAAs allow for simple click-chemistry derivatization, photo-cage control of enzyme activity, and site-specific placement of posttranslational modifications. We previously described a modular amber suppression plasmid system for generating stable cell lines via piggyBac transposition in a range of mammalian cells. Here we detail a general protocol for the generation of CRISPR-Cas9 knock-in cell lines using the same plasmid system. The knock-in strategy relies on CRISPR-Cas9-induced double-strand breaks (DSBs) and nonhomologous end joining (NHEJ) repair to target the PylT/RS expression cassette to the AAVS1 safe harbor locus in human cells. MmaPylRS expression from this single locus is sufficient for efficient amber suppression when the cells are subsequently transfected transiently with a PylT/gene of interest plasmid.

A neuronal cell-based reporter system for monitoring the activity of HDAC2.

Given that histone acetylation via histone acetyltransferases (HATs) and histone deacetylases (HDACs) is significant in memory formation, HDAC2 has been thoroughly investigated as a potential therapeutic target for the treatment of cognitive dysfunction. Although HDAC inhibitors have been discovered through in vitro enzyme assay, off-target effects on other HDACs are common due to their conserved catalytic domains. Each HDAC could be regulated by specific intracellular molecular mechanisms, raising the possibility that a cell-based assay could identify selective inhibitors targeting specific HDACs through their regulatory mechanisms. Here, we propose a versatile, cell-based reporter system for screening HDAC2 inhibitors. Through RNA-sequencing from human cultured neuronal cells, we determined that expression of a transcriptional repressor, inhibitor of DNA binding 1 (ID1), is increased by knockdown of HDAC2. We also established the knock-in neuronal cell lines of a bioluminescence reporter gene to ID1. The knock-in cell lines showed significant reporter activity by known HDAC inhibitors and by HDAC2-knockdown but not by HDAC1-knockdown. Thus, our neuronal cell-based reporter system is a promising method for screening the specific inhibitors of HDAC2 but not HDAC1, by potentially targeting not only HDAC2, but also the regulatory mechanisms of HDAC2 in neurons.