CRISPR-Cas9 System Research Articles

PtARgenOM-A Flexible Vector For CRISPR/CAS9 Nonviral Delivery.

Viral-mediated delivery of the CRISPR-Cas9 system is one the most commonly used techniques to modify the genome of a cell, with the aim of analyzing the function of the targeted gene product. While these approaches are rather straightforward for membrane-bound proteins, they can be laborious for intracellular proteins, given that selection of full knockout (KO) cells often requires the amplification of single-cell clones. Moreover, viral-mediated delivery systems, besides the Cas9 and gRNA, lead to the integration of unwanted genetic material, such as antibiotic resistance genes, introducing experimental biases. Here, an alternative non-viral delivery approach is presented for CRISPR/Cas9, allowing efficient and flexible selection of KO polyclonal cells. This all-in-one mammalian CRISPR-Cas9 expression vector, ptARgenOM, encodes the gRNA and the Cas9 linked to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein and the puromycin N-acetyltransferase, allowing for transient, expression-dependent selection and enrichment of isogenic KO cells. After evaluation using more than 12 distinct targets in 6 cell lines, ptARgenOM is found to be efficient in producing KO cells, reducing the time required to obtain a polyclonal isogenic cell line by 4-6 folds. Altogether ptARgenOM provides a simple, fast, and cost-effective delivery tool for genome editing.

50 Gene Editing Applications in Livestock Production

Abstract Gene editing is a technology that allows changes in the DNA of organisms from plants to animals, to humans. Its application in research has been around for a long time, and different techniques were developed over the years such as Zinc-finger nucleases, TALENs and more recently the CRISPR-Cas9 system. The latter has gained popularity for being much simpler to implement and at the same time being able to perform more precise edits with less off targets. Scientists and farmers have been using genetics selection to improve productivity and sustainability of agriculture for many decades using information from phenotypes, pedigrees, and more recently genomic information. With the increasing demand for food production, the investigation and adoption of new technologies will continue to drive and accelerate genetic improvement in animals and plants to meet consumer demands and expectations. A lot of effort has been devoted to developing gene editing research and applications for a variety of traits to improve production, health, and wellbeing of farmed animals. Although gene editing and more specifically the CRISPR technology was met with great expectations for genetic improvements in animals, a few challenges are still unresolved and are under constant research and development. For instance, many of the economically relevant traits for animal production are controlled by many genes with small effects (polygenic traits). For these traits, editing many loci would be required to make significant improvements, which adds a lot of complexity for the current technologies. Another challenge for further development, and deployment of this technology has been on the regulatory side, in which legislation for the use of gene edited animals for food production is still under development, and many regulatory aspects are not clear. These have been largely addressed on case-by-case basis, with recent approvals for Atlantic Salmon (fast growing), swine (hypoallergenic) and beef cattle (slick coat) being granted for specific use cases. Other examples of applications of gene editing are the coat color change, and creation of polled animals in cattle, which target improvements on environmental adaptability and animal welfare. In swine, researchers have been working on developing pigs that are resistant to the porcine reproductive and respiratory syndrome (PRRS) which could lead to a great health and economic benefits for the swine industry. These are a few examples of how gene editing could help improve disease resistance, well-being and climate adaptation traits for many livestock and aquaculture species in the future. Further refinements of the technology, the development of policies and legislation, and strategies for deployment in commercial applications will continue to be key aspects to guide a safe and responsible use of gene editing in livestock production.

Genome-scale targeted mutagenesis in Brassica napus using a pooled CRISPR library.

The recently constructed mutant libraries of diploid crops by the CRISPR-Cas9 system have provided abundant resources for functional genomics and crop breeding. However, because of the genome complexity, it is a big challenge to accomplish large-scale targeted mutagenesis in polyploid plants. Here, we demonstrate the feasibility of using a pooled CRISPR library to achieve genome-scale targeted editing in an allotetraploid crop of Brassica napus A total of 18,414 sgRNAs were designed to target 10,480 genes of interest, and afterward, 1104 regenerated transgenic plants harboring 1088 sgRNAs were obtained. Editing interrogation results revealed that 93 of the 178 genes were identified as mutated, thus representing an editing efficiency of 52.2%. Furthermore, we have discovered that Cas9-mediated DNA cleavages tend to occur at all the target sites guided by the same individual sgRNA, a novel finding in polyploid plants. Finally, we show the strong capability of reverse genetic screening for various traits with the postgenotyped plants. Several genes, which might dominate the fatty acid profile and seed oil content and have yet to be reported, were unveiled from the forward genetic studies. Our research provides valuable resources for functional genomics, elite crop breeding, and a good reference for high-throughput targeted mutagenesis in other polyploid plants.

Expression and localization of intermediate filament synemin isoforms in human pluripotent stem cells and their cardiac derivates

Synemin is an intermediate filament family involved in the cytoarchitecture of skeletal, cardiac and smooth muscle cells. In mammals, the SYNM gene encodes 3 isoforms: H (α, 170 kDa), M (β, 140 kDa) and L (Low, 41 kDa) through alternative splicing. In mouse, H and M isoform expression begins in the embryo while L isoform is only found in adult. Mouse embryonic pluripotent stem cells express M isoform as well as Oct-3/4 and SSEA-1. Two missense mutations have been recorded in patients with dilated cardiomyopathy and, α and β synemin levels increased in failing human hearts. Little is known about synemin expression in human induced pluripotent stem cells (hiPSC) and their cardiac derivatives. The aims are: (1) To characterize expression of synemin isoforms in hiPSC differentiation towards the cardiac lineage; (2) To analyze the impact of synemin in a human in vitro model of cardiac hypertrophy using hiPSC-derived cardiomyocytes (hiPSC-CMs). Expression of synemin isoforms was investigated in 2 lines of healthy donor-derived hiPSC and hiPSC-CMs by RT-qPCR, Western blot and immunofluorescent analysis. Synemin and α-actinin immunostaining was carried out on hiPSC-CMs treated 7 days with 200 nM AngII or 100 nM Iso following by an estimate of cell size and contraction measurements (ImageJ). Two different lines of healthy donor-derived hiPSC express the 3 isoforms of synemin at mRNA and protein levels. Synemin protein is detected in the cytoplasm and potentially in the nucleus of hiPSC expressing Nanog and Oct3/4. In hiPSC-CMs at 30 days of differentiation, expression of α, β and L isoforms increased with a fold change of 35, 80 and 114 respectively compared with hiPSC. Immunostaining reveals a cytoplasmic localization of synemin with striated pattern. An increase of synemin protein expression and nuclear localization was shown in hiPSC-CMs treated by hypertrophic agents. We showed for the first time that hiPSC and hiPSC-CMs express all known synemin isoforms. Moreover, subcellular localization of this protein change under hypertrophic conditions. Based on the literature, these data could suggest a potential role of synemin in cell motility, proliferation and also in the modulation of cardiac hypertrophy. To better understand these functions we have generated two synemin KO hiPSC lines using Dual-Guide CRISPR-Cas9 system of which characterization is ongoing.

Evaluation of a Novel FKS1 R1354H Mutation Associated with Caspofungin Resistance in Candida auris Using the CRISPR-Cas9 System.

Outbreaks of invasive infections, with high mortality rates, caused by multidrug-resistant Candida auris have been reported worldwide. Although hotspot mutations in FKS1 are an established cause of echinocandin resistance, the actual contribution of these mutations to echinocandin resistance remains unknown. Here, we sequenced the FKS1 gene of a caspofungin-resistant clinical isolate (clade I) and identified a novel resistance mutation (G4061A inducing R1354H). We applied the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to generate a recovered strain (H1354R) in which only this single nucleotide mutation was reverted to its wild-type sequence. We also generated mutant strains with only the R1354H mutation introduced into C. auris wild-type strains (clade I and II) and analyzed their antifungal susceptibility. Compared to their parental strains, the R1354H mutants exhibited a 4- to 16-fold increase in caspofungin minimum inhibitory concentration (MIC) while the H1354R reverted strain exhibited a 4-fold decrease in caspofungin MIC. In a mouse model of disseminated candidiasis, the in vivo therapeutic effect of caspofungin was more closely related to the FKS1 R1354H mutation and the virulence of the strain than its in vitro MIC. The CRISPR-Cas9 system could thus aid in elucidating the mechanism underlying drug resistance in C. auris.

The antioxidant protein glutaredoxin 1 is essential for oxidative stress response and pathogenicity of Toxoplasma gondii.

Glutaredoxins (Grxs) are ubiquitous antioxidant proteins involved in many molecular processes to protect cells against oxidative damage. Here, we study the roles of Grxs in the pathogenicity of Toxoplasma gondii. We show that Grxs are localized in the mitochondria (Grx1), cytoplasm (Grx2), and apicoplast (Grx3, Grx4), while Grx5 had an undetectable level of expression. We generated Δgrx1-5 mutants of T. gondii type I RH and type II Pru strains using CRISPR-Cas9 system. No significant differences in the infectivity were detected between four Δgrx (grx2-grx5) strains and their respective wild-type (WT) strains in vitro or in vivo. Additionally, no differences were detected in the production of reactive oxygen species, total antioxidant capacity, superoxide dismutase activity, and sensitivity to external oxidative stimuli. Interestingly, RHΔgrx1 or PruΔgrx1 exhibited significant differences in all the investigated aspects compared to the other grx2-grx5 mutant and WT strains. Transcriptome analysis suggests that deletion of grx1 altered the expression of genes involved in transport and metabolic pathways, signal transduction, translation, and obsolete oxidation-reduction process. The data support the conclusion that grx1 supports T. gondii resistance to oxidative killing and is essential for the parasite growth in cultured cells and pathogenicity in mice and that the active site CGFS motif was necessary for Grx1 activity.

Regulation of Hsa-miR-4639-5p expression and its potential role in the pathogenesis of Parkinson's disease.

Decreased DJ-1 protein impairs antioxidative activity of neurons and plays an important role in the occurrence of Parkinson's disease (PD). We have previously identified hsa-miR-4639-5p as the post-transcriptional regulator of DJ-1. Increased expression of hsa-miR-4639-5p reduced DJ-1 level and increased oxidative stress leading to neuronal death. Therefore, understanding the detailed mechanisms by which hsa-miR-4639-5p expression is regulated will not only facilitate diagnosis but also inform the pathogenesis of PD. We examined hsa-miR-4639-5 in either the plasma or exosomes derived from the central nervous system (CNS) neurons of PD patients and healthy controls. We showed that CNS-derived exosomes gave rise to the increased plasma hsa-miR-4639-5p in PD patients, pointing to hsa-miR-4639-5p dysregulation in the brain of PD patients. Using a dual-luciferase assay and a CRISPR-Cas9 system, we identified a core promoter of hsa-miR-4639 (-560 to -275 upstream the transcriptional starting site) of the gene for myosin regulatory light chain interacting protein. A polymorphism in the core promoter (rs760632 G>A) could enhance hsa-miR-4639-5p expression and increase PD risk. Furthermore, using MethylTarget™ assay, ChIP-qPCR, and specific inhibitors, we demonstrated that hsa-miR4639-5p expression was regulated by HDAC11-mediated histone acetylation but not DNA methylation/demethylation. Taken together, our study provides evidence that hsa-miR-4639-5p is a potential diagnostic marker and therapeutic target for PD. Interventions targeting hsa-miR-4639-5p might represent a novel therapy to promote healthy aging.

A novel prognostic signatures based on metastasis- and immune-related gene pairs for colorectal cancer.

Metastasis remains the leading cause of mortality in patients diagnosed with colorectal cancer (CRC). The pivotal contribution of the immune microenvironment in the initiation and progression of CRC metastasis has gained significant attention. A total of 453 CRC patients from The Cancer Genome Atlas (TCGA) were included as the training set, and GSE39582, GSE17536, GSE29621, GSE71187 were included as the validation set. The single-sample gene set enrichment analysis (ssGSEA) was performed to assess the immune infiltration of patients. Least absolute shrinkage and selection operator (LASSO) regression analysis, Time-dependent receiver operating characteristic (ROC) and Kaplan-Meier analysis were used to construct and validate risk models based on R package. CTSW and FABP4-knockout CRC cells were constructed via CRISPR-Cas9 system. Western-blot and Transwell assay were utilized to explore the role of fatty acid binding protein 4 (FABP4) / cathepsin W (CTSW) in CRC metastasis and immunity. Based on the normal/tumor, high-/low-immune cell infiltration, and metastatic/non-metastatic group, we identified 161 differentially expressed genes. After random assignment and LASSO regression analysis, a prognostic model containing 3 metastasis- and immune-related gene pairs was constructed and represented good prognostic prediction efficiency in the training set and 4 independent CRC cohorts. According to this model, we clustered patients and found that the high-risk group was associated with stage, T and M stage. In addition, the high-risk group also shown higher immune infiltration and high sensitivity to PARP inhibitors. Further, FABP4 and CTSW derived from the constitutive model were identified to be involved in metastasis and immunity of CRC. In conclusion, a validated prognosis predictive model for CRC was constructed. CTSW and FABP4 are potential targets for CRC treatment.

Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering.

Non-coding RNAs (crRNAs) produced from clustered regularly interspaced short palindromic repeats (CRISPR) loci and CRISPR-associated (Cas) proteins of the prokaryotic CRISPR-Cas systems form complexes that interfere with the spread of transmissible genetic elements through Cas-catalysed cleavage of foreign genetic material matching the guide crRNA sequences. The easily programmable targeting of nucleic acids enabled by these ribonucleoproteins has facilitated the implementation of CRISPR-based molecular biology tools for in vivo and in vitro modification of DNA and RNA targets. Despite the diversity of DNA-targeting Cas nucleases so far identified, native and engineered derivatives of the Streptococcus pyogenes SpCas9 are the most widely used for genome engineering, at least in part due to their catalytic robustness and the requirement of an exceptionally short motif (5'-NGG-3' PAM) flanking the target sequence. However, the large size of the SpCas9 variants impairs the delivery of the tool to eukaryotic cells and smaller alternatives are desirable. Here, we identify in a metagenome a new CRISPR-Cas9 system associated with a smaller Cas9 protein (EHCas9) that targets DNA sequences flanked by 5'-NGG-3' PAMs. We develop a simplified EHCas9 tool that specifically cleaves DNA targets and is functional for genome editing applications in prokaryotes and eukaryotic cells.

Design of high-monounsaturated fatty acid soybean seed oil using GmPDCTs knockout via a CRISPR-Cas9 system.

Design of high-monounsaturated fatty acid soybean seed oil using GmPDCTs knockout via a CRISPR-Cas9 system.

A prime editor efficiently repaired human induced pluripotent stem cells with AR gene mutation (c.2710G > A; p. V904M)

Prime Editor (PE) is a precise genome manipulation technology based on the CRISPR-Cas9 system, while its application in human induced pluripotent stem cells (iPSCs) remains limited. Here, we established a repaired hiPS cell line (SKLRMi001-A-1) from hiPSCs with androgen receptor (AR) mutation (c.2710G > A; p.V904M). The repaired iPSC line expressed pluripotency markers, retained normal karyotype, showed the capability of differentiating into three germ layers and was absence of mycoplasma infection. The repaired iPSC line will help to elucidate the mechanism of androgen insensitivity syndrome (AIS) and benefit treatment for AIS in the future.

Photocleavable Ortho-Nitrobenzyl-Protected DNA Architectures and Their Applications.

This review article introduces mechanistic aspects and applications of photochemically deprotected ortho-nitrobenzyl (ONB)-functionalized nucleic acids and their impact on diverse research fields including DNA nanotechnology and materials chemistry, biological chemistry, and systems chemistry. Specific topics addressed include the synthesis of the ONB-modified nucleic acids, the mechanisms involved in the photochemical deprotection of the ONB units, and the photophysical and chemical means to tune the irradiation wavelength required for the photodeprotection process. Principles to activate ONB-caged nanostructures, ONB-protected DNAzymes and aptamer frameworks are introduced. Specifically, the use of ONB-protected nucleic acids for the phototriggered spatiotemporal amplified sensing and imaging of intracellular mRNAs at the single-cell level are addressed, and control over transcription machineries, protein translation and spatiotemporal silencing of gene expression by ONB-deprotected nucleic acids are demonstrated. In addition, photodeprotection of ONB-modified nucleic acids finds important applications in controlling material properties and functions. These are introduced by the phototriggered fusion of ONB nucleic acid functionalized liposomes as models for cell-cell fusion, the light-stimulated fusion of ONB nucleic acid functionalized drug-loaded liposomes with cells for therapeutic applications, and the photolithographic patterning of ONB nucleic acid-modified interfaces. Particularly, the photolithographic control of the stiffness of membrane-like interfaces for the guided patterned growth of cells is realized. Moreover, ONB-functionalized microcapsules act as light-responsive carriers for the controlled release of drugs, and ONB-modified DNA origami frameworks act as mechanical devices or stimuli-responsive containments for the operation of DNA machineries such as the CRISPR-Cas9 system. The future challenges and potential applications of photoprotected DNA structures are discussed.

Validation of CRISPR activation system in Aedes cells using multicistronic plasmid vectors.

Aedes mosquitoes transmit several pathogens including flaviviruses to humans which result in high morbidity and mortality. Owing to adaptability and climate change, these mosquito vectors are predicted to establish in new geographical areas thus exposing larger populations to the risk of infection. Therefore, control of Aedes vector is necessary to prevent disease transmission. Recently, genetic approaches to vector control have shown promise; however, the tools and methods for manipulating the mosquito genome are rather limited. While CRISPR-Cas9 system has been adapted for gene editing purposes in Aedes mosquito, the dCas9-based transcription control of genes remain unexplored. In this study we report implementation of the CRISPR activation system in Aedes cells. For this we designed, constructed and tested a bi-partite plasmid-based strategy that allows expression of the dCas9-VPR and targeting guide RNA together with a reporter cassette. Quantitative analysis of the fluorescent reporter gene levels showed a robust over-expression validating CRISPR activation in Aedes cells. This strategy and the biological parts will be useful resource for synthetic transcription factor-based robust upregulation of Aedes genes for application of synthetic biology approaches for vector control.

A CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness

The lack of preparedness for detecting and responding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen (i.e., COVID-19) has caused enormous harm to public health and the economy. Testing strategies deployed on a population scale at day zero, i.e., the time of the first reported case, would be of significant value. Next-generation sequencing (NGS) has such capabilities; however, it has limited detection sensitivity for low-copy-number pathogens. Here, we leverage the CRISPR-Cas9 system to effectively remove abundant sequences not contributing to pathogen detection and show that NGS detection sensitivity of SARS-CoV-2 approaches that of RT-qPCR. The resulting sequence data can also be used for variant strain typing, co-infection detection, and individual human host response assessment, all in a single molecular and analysis workflow. This NGS work flow is pathogen agnostic and, therefore, has the potential to transform how large-scale pandemic response and focused clinical infectious disease testing are pursued in the future.

Transformer-based anti-noise models for CRISPR-Cas9 off-target activities prediction.

The off-target effect occurring in the CRISPR-Cas9 system has been a challenging problem for the practical application of this gene editing technology. In recent years, various prediction models have been proposed to predict potential off-target activities. However, most of the existing prediction methods do not fully exploit guide RNA (gRNA) and DNA sequence pair information effectively. In addition, available prediction methods usually ignore the noise effect in original off-target datasets. To address these issues, we design a novel coding scheme, which considers the key features of mismatch type, mismatch location and the gRNA-DNA sequence pair information. Furthermore, a transformer-based anti-noise model called CrisprDNT is developed to solve the noise problem that exists in the off-target data. Experimental results of eight existing datasets demonstrate that the method with the inclusion of the anti-noise loss functions is superior to available state-of-the-art prediction methods. CrisprDNT is available at https://github.com/gzrgzx/CrisprDNT.

Overcoming the Limitations of CRISPR-Cas9 Systems in Saccharomyces cerevisiae: Off-Target Effects, Epigenome, and Mitochondrial Editing.

Modification of the genome of the yeast Saccharomyces cerevisiae has great potential for application in biological research and biotechnological advancements, and the CRISPR-Cas9 system has been increasingly employed for these purposes. The CRISPR-Cas9 system enables the precise and simultaneous modification of any genomic region of the yeast to a desired sequence by altering only a 20-nucleotide sequence within the guide RNA expression constructs. However, the conventional CRISPR-Cas9 system has several limitations. In this review, we describe the methods that were developed to overcome these limitations using yeast cells. We focus on three types of developments: reducing the frequency of unintended editing to both non-target and target sequences in the genome, inducing desired changes in the epigenetic state of the target region, and challenging the expansion of the CRISPR-Cas9 system to edit genomes within intracellular organelles such as mitochondria. These developments using yeast cells to overcome the limitations of the CRISPR-Cas9 system are a key factor driving the advancement of the field of genome editing.

Abstract LB357: CRISPR-Cas9 targeting of somatic mutations selectively kills cancer cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) accounts for 3% of all cancers but is the 3rd leading cause of cancer death in the United States. This staggering discrepancy is partly because most pancreatic cancers evade early detection and patients most commonly present with metastatic disease. As cancer initiates from a single transformed cell, passenger mutations that accumulate over the course of the patient’s lifetime in that cell are then clonally propagated to the growing tumor and metastases, providing a targetable molecular signature throughout the cancer. Here we propose a CRISPR-Cas9 gene therapy aimed at these tumor-specific somatic mutations. Using whole genome sequencing of rapid autopsy tumor and normal samples from five patient cases, we identified single base substitutions present in tumor tissues and absent in patient-matched normal tissue. A subset (6.6% on average) of these mutations lead to novel protospacer adjacent motif (PAM) sites. These tumor specific PAM sites are necessary for Cas9 binding and DNA scission and are therefore selectively targetable by CRISPR-Cas9 systems. Using a hybrid capture/NGS approach, we demonstrate in patients with multiple metastases that the majority of these novel PAM sites, ~70%, are maintained among the different metastases. Using multitarget sgRNA which cut at a known number of sites in the human genome, we determined a relationship between number of CRISPR-Cas9 cuts and growth inhibition in PDAC cells. We found that 8-10 cut sites in non-coding regions are sufficient to induce >95% cancer cell death. Finally, using cocultures of fluorescently labeled PDAC cells and a multiplexed sgRNA array targeting multiple tumor specific PAMs, we demonstrate selective cell killing of the targeted cell line. Importantly, deep sequencing of patient-matched normal lymphocytes lacking the tumor-specific PAMs treated with the same sgRNA array exhibited no editing at the targeted loci. This work establishes a bioinformatic platform for the discovery of tumor-specific CRISPR targets in the context of metastatic disease and demonstrates selective killing to cancer cells while sparing the patient’s normal cells. These data establish the proof of principle for a gene therapy approach targeting metastatic disease. Citation Format: Kirsten Bowland, Selina Shiqing K. Teh, Alyza Skaist, Ralph H. Hruban, Nicholas J. Roberts, Chrisine Iacobuzio-Donahue, James R. Eshleman. CRISPR-Cas9 targeting of somatic mutations selectively kills cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB357.

Mitoxantrone and Mitoxantrone-Loaded Iron Oxide Nanoparticles Induce Cell Death in Human Pancreatic Ductal Adenocarcinoma Cell Spheroids

Pancreatic ductal adenocarcinoma is a hard-to-treat, deadly malignancy. Traditional treatments, such as surgery, radiation and chemotherapy, unfortunately are still not able to significantly improve long-term survival. Three-dimensional (3D) cell cultures might be a platform to study new drug types in a highly reproducible, resource-saving model within a relevant pathophysiological cellular microenvironment. We used a 3D culture of human pancreatic ductal adenocarcinoma cell lines to investigate a potential new treatment approach using superparamagnetic iron oxide nanoparticles (SPIONs) as a drug delivery system for mitoxantrone (MTO), a chemotherapeutic agent. We established a PaCa DD183 cell line and generated PANC-1SMAD4 (-/-) cells by using the CRISPR-Cas9 system, differing in a prognostically relevant mutation in the TGF-β pathway. Afterwards, we formed spheroids using PaCa DD183, PANC-1 and PANC-1SMAD4 (-/-) cells, and analyzed the uptake and cytotoxic effect of free MTO and MTO-loaded SPIONs by microscopy and flow cytometry. MTO and SPION-MTO-induced cell death in all tumor spheroids in a dose-dependent manner. Interestingly, spheroids with a SMAD4 mutation showed an increased uptake of MTO and SPION-MTO, while at the same time being more resistant to the cytotoxic effects of the chemotherapeutic agents. MTO-loaded SPIONs, with their ability for magnetic drug targeting, could be a future approach for treating pancreatic ductal adenocarcinomas.

Abstract 6208: Targeting DNA polymerase theta and ATM leads to synergistic killing of mantle cell lymphoma cells

Abstract Introduction: Rapid cell proliferation requires intact and faithful DNA damage repair mechanisms. DNA polymerase theta (POLQ) plays a key role in repairing DNA double-strand breaks through the microhomology-mediated end-joining (MMEJ), which is one of the three main pathways involved in repairing replication-induced double-strand breaks. Limited data have suggested that concurrent depletion of POLQ and ataxia-telangiectasia mutated (ATM) could be embryonic lethal. Hence, this phenomenon has the potential to be exploited for therapeutic benefit in cancers where ATM mutations are commonly seen. Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma marked by (11;14) translocation with ATM alterations seen in 40-50% of patients. There is an unmet need to find novel therapeutic strategies, especially in relapsed and/or refractory (R/R) MCL. Here, we investigated whether targeting POLQ and ATM could be a potential therapeutic strategy in MCL. Methods: In vitro studies were conducted by using MCL cell lines. CRISPR-Cas9 system was used to genetically deplete POLQ and ATM genes and sgRNAs co-expressing fluorescence markers were used to track the cell population with respective genotypes over time. Cell viability was assessed by CellTiter-Glo assay and flow cytometry. All cell lines were profiled for ATM expression and activity. A p-value of < 0.05 was considered statistically significant. The combination index of <1 was defined as synergistic. Results: CRISPR-Cas9-mediated depletion of POLQ significantly decreased cell proliferation in multiple MCL cell lines. In particular, Granta-519, which possesses a single copy of kinase-dead ATM that is reduced in expression, was most sensitive to POLQ depletion. Concurrent genetic depletion of ATM and POLQ resulted in a synergistic antiproliferative effect in ATM-proficient MCL cell lines. Subsequently, this cellular phenotype caused by the genetic intervention was recapitulated by using two POLQ inhibitors (novobiocin and ART558) and an ATM inhibitor (AZD0156). In vitro, single-agent treatment with novobiocin or ART558 caused a significant cytotoxic effect at physiologically relevant concentrations in ATM-deficient cells and co-treatment of novobiocin or ART558 with AZD0156 was synergistic in killing ATM-proficient MCL cells. Importantly, POLQ inhibitors significantly decreased the cell viability of MCIR1, which is an ibrutinib-resistant MCL cell line. Mechanistically, novobiocin or ART558 treatment induced gH2AX and cleaved PARP upregulation, which was further enhanced by ATM depletion, suggesting that co-inhibition of POLQ and ATM caused apoptosis due to the accumulation of unrepaired DNA damage. Conclusion: POLQ is a promising target in MCL, especially in ATM-deficient setting. In ATM-proficient MCL, targeting ATM and POLQ is synergistic. Our data has the potential to uncover novel biomarker-driven drug therapy of POLQ inhibitors in R/R MCL. Citation Format: Jithma Prasad Abeykoon, Shuhei Asada, Kalindi Parmar, Xiaosheng Wu, Thomas Witzig, Geoffrey Shapiro, Alan D. D'Andrea. Targeting DNA polymerase theta and ATM leads to synergistic killing of mantle cell lymphoma cells. [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 6208.

Abstract 29: Characterization of a murine intestinal cancer homograft model for therapeutic evaluation

Abstract Introduction: Intestinal cancers are one of the most lethal malignancies and are responsible for a mass of cancer-related deaths. The discovery of new drugs and therapeutic strategies is underway, and to complement this, the development of superior animal models is required for preclinical studies. Genetically engineered mouse models (GEMMs) mimic de novo human tumor growth within an intact tumor microenvironment and are potent immunocompetent models for studying pathogenesis and immuno-oncology of intestinal cancers. GEMMs with dysfunctional DNA mismatch repair (MMR) genes, like MHL1 and MSH2, manifest intestinal tumors related to microsatellite instability (MSI). However, the low incidence and distinct latency among individuals hamper the application of these GEMMs in preclinical research. In this study, a murine intestinal cancer homograft from GEMMs with MMR deficiency is established to enroll larger cohorts of mice for preclinical studies. Methods: MMR-deficient GEMMs were generated by knockout of Mlh1 gene with a CRISPR-Cas9 system. Intestinal cancer development was investigated in 16 GEMMs. The developed tumor in the intestine of GEMM was isolated by surgery and inoculated into C57BL/6 mice subcutaneously to establish a murine homograft model. The tumor homograft was passaged and expanded in vivo, and stable tumor growth was established over consecutive passages. Histopathological studies were conducted for both the original and passaged tumors. Genomic mutations and gene expression were analyzed by whole exome sequencing and RNA-sequencing, and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. Therapeutic efficacy of chemotherapies and immune checkpoint inhibitors were evaluated in vivo for tumor growth inhibition. Results: The intestinal tumors or lesions were observed in two of the sixteen Mlh1-KO mice with an incidence of 12.5%. A murine homograft model of small bowel adenocarcinoma was successfully established using the tumor located in jejunum of a Mlh1-KO mouse. This model was stably passaged in vivo with semblable pathological features as the original lesion in Mlh1-KO mice. The various degrees of TILs and response to treatments revealed the potential of this model for in vivo investigation of immune response. Conclusion: Our murine intestinal cancer homograft model presented rapid and stable subcutaneous growth in immunocompetent mice, providing an efficient approach for intestinal cancer researchers and a superior preclinical model for new drug discovery. Citation Format: Jinxi Wang, Leilei Chen, Likun Zhang, Jingqian Zhu, Ludovic Bourre, Jingjing Wang. Characterization of a murine intestinal cancer homograft model for therapeutic evaluation [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 29.