Liver cancer development driven by the AP-1/c-Jun~Fra-2 dimer through c-Myc.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. HCC incidence is on the rise, while treatment options remain limited. Thus, a better understanding of the molecular pathways involved in HCC development has become a priority to guide future therapies. While previous studies implicated the Activator Protein-1 (AP-1) (Fos/Jun) transcription factor family members c-Fos and c-Jun in HCC formation, the contribution of Fos-related antigens (Fra-) 1 and 2 is unknown. Here, we show that hepatocyte-restricted expression of a single chain c-Jun~Fra-2 protein, which functionally mimics the c-Jun/Fra-2 AP-1 dimer, results in spontaneous HCC formation in c-Jun~Fra-2hep mice. Several hallmarks of human HCC, such as cell cycle dysregulation and the expression of HCC markers are observed in liver tumors arising in c-Jun~Fra-2hep mice. Tumorigenesis occurs in the context of mild inflammation, low-grade fibrosis, and Pparγ-driven dyslipidemia. Subsequent analyses revealed increased expression of c-Myc, evidently under direct regulation by AP-1 through a conserved distal 3' enhancer. Importantly, c-Jun~Fra-2-induced tumors revert upon switching off transgene expression, suggesting oncogene addiction to the c-Jun~Fra-2 transgene. Tumors escaping reversion maintained c-Myc and c-Myc target gene expression, likely due to increased c-Fos. Interfering with c-Myc in established tumors using the Bromodomain and Extra-Terminal motif inhibitor JQ-1 diminished liver tumor growth in c-Jun~Fra-2 mutant mice. Thus, our data establish c-Jun~Fra-2hep mice as a model to study liver tumorigenesis and identify the c-Jun/Fra-2-Myc interaction as a potential target to improve HCC patient stratification and/or therapy.

CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression.

Metastatic prostate cancer (PCa) poses a significant therapeutic challenge with high mortality rates. Utilizing CRISPR-Cas9 in vivo, we target five potential tumor suppressor genes (Pten, Trp53, Rb1, Stk11, and RnaseL) in the mouse prostate, reaching humane endpoint after eight weeks without metastasis. By further depleting three epigenetic factors (Kmt2c, Kmt2d, and Zbtb16), lung metastases are present in all mice. While whole genome sequencing reveals few mutations in coding sequence, RNA sequencing shows significant dysregulation, especially in a conserved genomic region at chr5qE1 regulated by KMT2C. Depleting Odam and Cabs1 in this region prevents metastasis. Notably, the gene expression signatures, resulting from our study, predict progression-free and overall survival and distinguish primary and metastatic human prostate cancer. This study emphasizes positive genetic interactions between classical tumor suppressor genes and epigenetic modulators in metastatic PCa progression, offering insights into potential treatments.

STING activation counters glioblastoma by vascular alteration and immune surveillance.

Glioblastoma (GBM) is an aggressive brain tumor with a median survival of 15 months and has limited treatment options. Immunotherapy with checkpoint inhibitors has shown minimal efficacy in combating GBM, and large clinical trials have failed. New immunotherapy approaches and a deeper understanding of immune surveillance of GBM are needed to advance treatment options for this devastating disease. In this study, we used two preclinical models of GBM: orthotopically delivering either GBM stem cells or employing CRISPR-mediated tumorigenesis by adeno-associated virus, to establish immunologically proficient and non-inflamed tumors, respectively. After tumor development, the innate immune system was activated through long-term STING activation by a pharmacological agonist, which reduced tumor progression and prolonged survival. Recruitment and activation of cytotoxic T-cells were detected in the tumors, and T-cell specificity towards the cancer cells was observed. Interestingly, prolonged STING activation altered the tumor vasculature, inducing hypoxia and activation of VEGFR, as measured by a kinome array and VEGF expression. Combination treatment with anti-PD1 did not provide a synergistic effect, indicating that STING activation alone is sufficient to activate immune surveillance and hinder tumor development through vascular disruption. These results guide future studies to refine innate immune activation as a treatment approach for GBM, in combination with anti-VEGF to impede tumor progression and induce an immunological response against the tumor.

Pre-Clinical Models to Study Human Prostate Cancer.

Prostate cancer is a common cancer among men and typically progresses slowly for several decades before becoming aggressive and spreading to other organs, leaving few treatment options. While large animals have been studied, the dog's prostate is anatomically similar to humans and has been used to study spontaneous prostate cancer. However, most research currently focuses on the mouse as a model organism due to the ability to genetically modify their prostatic tissues for molecular analysis. One milestone in this research was the identification of the prostate-specific promoter Probasin, which allowed for the prostate-specific expression of transgenes. This has led to the generation of mice with aggressive prostatic tumors through overexpression of the SV40 oncogene. The Probasin promoter is also used to drive Cre expression and has allowed researchers to generate prostate-specific loss-of-function studies. Another landmark moment in the process of modeling prostate cancer in mice was the orthoptic delivery of viral particles. This technology allows the selective overexpression of oncogenes from lentivirus or the use of CRISPR to generate complex loss-of-function studies. These genetically modified models are complemented by classical xenografts of human prostate tumor cells in immune-deficient mice. Overall, pre-clinical models have provided a portfolio of model systems to study and address complex mechanisms in prostate cancer for improved treatment options. This review will focus on the advances in each technique.

Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of CRISPR/Cas ribonucleoprotein complexes supporting base editing, prime editing and in vivo gene modification.

Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks.

ER stress induces caspase-2-tBID-GSDME-dependent cell death in neurons lytically infected with herpes simplex virus type 2.

Neurotropic viruses, including herpes simplex virus (HSV) types 1 and 2, have the capacity to infect neurons and can cause severe diseases. This is associated with neuronal cell death, which may contribute to morbidity or even mortality if the infection is not controlled. However, the mechanistic details of HSV-induced neuronal cell death remain enigmatic. Here, we report that lytic HSV-2 infection of human neuron-like SH-SY5Y cells and primary human and murine brain cells leads to cell death mediated by gasdermin E (GSDME). HSV-2-induced GSDME-mediated cell death occurs downstream of replication-induced endoplasmic reticulum stress driven by inositol-requiring kinase 1α (IRE1α), leading to activation of caspase-2, cleavage of the pro-apoptotic protein BH3-interacting domain death agonist (BID), and mitochondria-dependent activation of caspase-3. Finally, necrotic neurons released alarmins, which activated inflammatory responses in human iPSC-derived microglia. In conclusion, lytic HSV infection in neurons activates an ER stress-driven pathway to execute GSDME-mediated cell death and promote inflammation.

Application of CRISPR for In Vivo Mouse Cancer Studies.

Clustered regularly interspaced short palindromic repeats (CRISPR) are widely used in cancer research to edit specific genes and study their functions. This applies both to in vitro and in vivo studies where CRISPR technology has accelerated the generation of specific loss- or gain-of-function mutations. This review focuses on CRISPR for generating in vivo models of cancer by editing somatic cells in specific organs. The delivery of CRISPR/Cas to designated tissues and specific cell compartments is discussed with a focus on different methods and their advantages. One advantage of CRISPR/Cas is the possibility to target multiple genes simultaneously in the same cell and therefore generate complex mutation profiles. This complexity challenges the interpretation of results and different methods to analyze the samples discussed herein. CRISPR-induced tumors are also different from classical tumors in pre-clinical models. Especially the clonal evolution of CRISPR-induced tumors adds new insight into cancer biology. Finally, the review discusses future perspectives for CRISPR technology in pre-clinical models with a focus on in vivo screening, CRISPR activation/inhibition, and the development of prime/ base-editing for the introduction of specific gene editing.

In Vivo Application of CRISPR/Cas9 Revealed Implication of Foxa1 and Foxp1 in Prostate Cancer Proliferation and Epithelial Plasticity.

Prostate cancer is the most common cancer in men in the Western world and the number is rising. Prostate cancer is notoriously heterogeneous, which makes it hard to generate and study in pre-clinical models. The family of Forkhead box (FOX) transcription factors are often altered in prostate cancer with especially high mutation burden in FOXA1 and FOXP1. FOXA1 harbors loss or gain of function mutations in 8% of prostate cancer, which increases to 14% in metastatic samples. FOXP1 predominately occurs with loss of function mutations in 7% of primary tumors, and similar incidents are found in metastatic samples. Here, we applied in vivo CRISPR editing, to study the loss of functions of these two FOX transcription factors, in murine prostate in combination with loss of Pten. Deficiency of Foxp1 increased proliferation in combination with loss of Pten. In contrast, proliferation was unchanged when androgen was deprived. The expression of Tmprss2 was increased when Foxp1 was mutated in vivo, showing that Foxp1 is a repressor for this androgen-regulated target. Furthermore, analysis of FOXP1 and TMPRSS2 expression in a human prostate cancer data set revealed a negative correlation. Mutation of Foxa1 in the murine prostate induces cell plasticity to luminal cells. Here, epithelial cells with loss of Foxa1 were transdifferentiated to cells with expression of the basal markers Ck5 and p63. Interestingly, these cells were located in the lumen and did not co-express Ck8. Overall, this study reveals that loss of Foxp1 increases cell proliferation, whereas loss of Foxa1 induces epithelial plasticity in prostate cancer.

A truncated reverse transcriptase enhances prime editing by split AAV vectors.

Prime editing is a new CRISPR-based, genome-editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with adeno-associated virus (AAV) viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4-fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. We also show that this minimized PE gives rise to superior lentiviral vector titers (46-fold) over the regular PE in an all-in-one PE lentiviral vector. We finally deliver the minimized PE to mouse liver by dual AAV8 vectors and show up to 6% precise editing of the PCSK9 gene, thereby demonstrating the value of this truncated split PE system for in vivo applications.

Treatment Represents a Key Driver of Metastatic Cancer Evolution.

Metastasis is the main cause of cancer death, yet the evolutionary processes behind it remain largely unknown. Here, through analysis of large panel-based genomic datasets from the AACR Genomics Evidence Neoplasia Information Exchange project, including 40,979 primary and metastatic tumors across 25 distinct cancer types, we explore how the evolutionary pressure of cancer metastasis shapes the selection of genomic drivers of cancer. The most commonly affected genes were TP53, MYC, and CDKN2A, with no specific pattern associated with metastatic disease. This suggests that, on a driver mutation level, the selective pressure operating in primary and metastatic tumors is similar. The most highly enriched individual driver mutations in metastatic tumors were mutations known to drive resistance to hormone therapies in breast and prostate cancer (ESR1 and AR), anti-EGFR therapy in non-small cell lung cancer (EGFR T790M), and imatinib in gastrointestinal cancer (KIT V654A). Specific mutational signatures were also associated with treatment in three cancer types, supporting clonal selection following anticancer therapy. Overall, this implies that initial acquisition of driver mutations is predominantly shaped by the tissue of origin, where specific mutations define the developing primary tumor and drive growth, immune escape, and tolerance to chromosomal instability. However, acquisition of driver mutations that contribute to metastatic disease is less specific, with the main genomic drivers of metastatic cancer evolution associating with resistance to therapy. SIGNIFICANCE: This study leverages large datasets to investigate the evolutionary landscape of established cancer genes to shed new light upon the mystery of cancer dissemination and expand the understanding of metastatic cancer biology.

Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic.

Type I interferons (IFN-I) play a critical role in human antiviral immunity, as demonstrated by the exceptionally rare deleterious variants of IFNAR1 or IFNAR2. We investigated five children from Greenland, Canada, and Alaska presenting with viral diseases, including life-threatening COVID-19 or influenza, in addition to meningoencephalitis and/or hemophagocytic lymphohistiocytosis following live-attenuated viral vaccination. The affected individuals bore the same homozygous IFNAR2 c.157T>C, p.Ser53Pro missense variant. Although absent from reference databases, p.Ser53Pro occurred with a minor allele frequency of 0.034 in their Inuit ancestry. The serine to proline substitution prevented cell surface expression of IFNAR2 protein, small amounts of which persisted intracellularly in an aberrantly glycosylated state. Cells exclusively expressing the p.Ser53Pro variant lacked responses to recombinant IFN-I and displayed heightened vulnerability to multiple viruses in vitro-a phenotype rescued by wild-type IFNAR2 complementation. This novel form of autosomal recessive IFNAR2 deficiency reinforces the essential role of IFN-I in viral immunity. Further studies are warranted to assess the need for population screening.

Three-dimensional culture models to study glioblastoma - current trends and future perspectives.

Glioblastoma (GBM) is the most prevalent form of primary malignant brain tumor in adults and remains almost invariably lethal owing to its aggressive and invasive nature. There have only been marginal improvements in its bleak survival rate of 12-15 months over the last four decades. The lack of preclinical models that efficiently recapitulate tumor biology and the tumor microenvironment is also in part responsible for the slow phase of translational GBM research. Emerging three-dimensional (3D) organoids and cell culture systems offer new and innovative possibilities for GBM modelling. These 3D models find their application to engineer the disease, screen drugs, establishing live biobank, and explore personalized therapy. Furthermore, these models can also be genetically modified by using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, which would allow one to study the specific role of key genes associated with gliomagenesis. Establishment of a coculture system with GBM cells to understand its invasive behavior is yet another major application of this model. Despite these merits, the organoid models also have certain limitations, including the absence of immune responses and vascular systems. In recent years, major progress has been made in the development and refinement of 3D models of GBM. In this review, we intend to highlight these recent advances and the potential future implications of this rapidly evolving field, which should facilitate a better understanding of GBM biology.

Targeting AP-1 transcription factors by CRISPR in the prostate.

Prostate cancer is the second most diagnosed cancer in men. It is a slow progressing cancer, but when the disease reaches an advanced stage, treatment options are limited. Sequencing analyses of cancer samples have identified genes that can potentially drive disease progression. We implemented the CRISPR/Cas9 technology to simultaneously manipulate multiple genes in the murine prostate and thus to functionally test putative cancer driver genes in vivo. The activating protein-1 (AP-1) transcription factor is associated with many different cancer types, with the proto-oncogenes JUN and FOS being the two most intensely studied subunits. We analyzed expression of FOS and JUNB in human prostate cancer datasets and observed decreased expression in advanced stages. By applying CRISPR/Cas9 technology, the role of these two transcription factors in prostate cancer progression was functionally tested. Our data revealed that loss of either JunB or Fos in the context of Pten loss drives prostate cancer progression to invasive disease. Furthermore, loss of Fos increases Jun expression, and CRISPR inactivation of Jun in this context decreases cell proliferation. Overall, these in vivo studies reveal that JunB and Fos exhibit a tumor suppressor function by repressing invasive disease, whereas Jun is oncogenic and increases cell proliferation. This demonstrates that AP-1 factors are implicated in prostate cancer progression at different stages and display a dual function as tumor suppressor and as an oncogene in cancer progression.

In vivo CRISPR inactivation of Fos promotes prostate cancer progression by altering the associated AP-1 subunit Jun.

Prostate cancer is a major global health concern with limited treatment options for advanced disease. Its heterogeneity challenges the identification of crucial driver genes implicated in disease progression. Activating protein-1 (AP-1) transcription factor is associated with cancer since the first identification of its subunits, the proto-oncogenes JUN and FOS. Whereas both JUN and FOS have been implicated in prostate cancer, this study provides the first functional evidence that FOS acts as a tumor suppressor during prostate cancer progression and invasion. Data mining revealed decreased FOS expression in prostate cancer and a further downregulation in metastatic disease, consistent with FOS expression in cell lines derived from different prostate cancer stages. FOS deficiency in prostate cancer cell lines increases cell proliferation and induces oncogenic pathway alterations. Importantly, in vivo CRISPR/Cas9-mediated Fos and Pten double mutation in murine prostate epithelium results in increased proliferation and invasiveness compared to the abrogation of Pten alone. Interestingly, enhanced Jun expression is observed in the murine prostatic intraepithelial neoplasia lacking Fos. CRISPR/Cas9-mediated knockout of Jun combined with Fos and Pten deficiency diminishes the increased proliferation rate in vivo but not the ability to form invasive disease. Overall, we demonstrate that loss of Fos promotes disease progression from clinical latent prostate cancer to advanced disease through accelerated proliferation and invasiveness, partly through Jun.

Comparative Analysis of Stk11/Lkb1 versus Pten Deficiency in Lung Adenocarcinoma Induced by CRISPR/Cas9.

This study focused on STK11, PTEN, KRAS, and TP53, which are often found to be mutated in lung cancer. We compared Stk11 and Pten implication in lung cancer in combination with loss of Trp53 and gain of function of Kras in a CRISPR/Cas9 mouse model. Mice with loss of Stk11, Trp53, and KrasG12D mutation (SKT) reached human endpoint at around four months post-initiation. In comparison, mice with loss of Pten, Trp53, and KrasG12D mutation (PKT) survived six months or longer post-initiation. Pathological examination revealed an increase in proliferation in SKT deficient lung epithelia compared to PKT. This difference was independent of Pten loss, indicating that loss of Pten is dispensable for cell proliferation in lung adenocarcinoma. Furthermore, tumors with loss of Stk11, Trp53, and KrasG12D mutation had a significantly higher progression rate, monitored by PET/MRI scanning, compared to mice with loss of Pten, Trp53, and KrasG12D mutation, revealing that mutations in Stk11 are essential for adenocarcinoma progression. Overall, by using the CRISPR/Cas9 mouse model of lung adenocarcinoma, we showed that mutations in Stk11 are a key driver, whereas loss of Pten is dispensable for adenocarcinoma progression.

Brain immune cells undergo cGAS/STING-dependent apoptosis during herpes simplex virus type 1 infection to limit type I IFN production.

Protection of the brain from viral infections involves the type I IFN (IFN-I) system, defects in which render humans susceptible to herpes simplex encephalitis (HSE). However, excessive cerebral IFN-I levels lead to pathologies, suggesting the need for tight regulation of responses. Based on data from mouse models, human HSE cases, and primary cell culture systems, we showed that microglia and other immune cells undergo apoptosis in the HSV-1-infected brain through a mechanism dependent on the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, but independent of IFN-I. HSV-1 infection of microglia induced cGAS-dependent apoptosis at high viral doses, whereas lower viral doses led to IFN-I responses. Importantly, inhibition of caspase activity prevented microglial cell death and augmented IFN-I responses. Accordingly, HSV-1-infected organotypic brain slices or mice treated with a caspase inhibitor exhibited lower viral load and an improved infection outcome. Collectively, we identify an activation-induced apoptosis program in brain immune cells that downmodulates local immune responses.

Author Correction: STEEP mediates STING ER exit and activation of signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

STEEP mediates STING ER exit and activation of signaling.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.

The cGAS-STING pathway is a therapeutic target in a preclinical model of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer, and the incidence of HCC is increasing. Recently, cancer immunotherapy has emerged as an efficient treatment against some cancers. Here we have used a mouse model of mutagen-induced HCC to explore the therapeutic usefulness of targeting the DNA-activated STING pathway in HCC. STING-deficient mice exhibited unaltered initial development of HCC, but had higher number of large tumors at late stages of disease. In the liver of STING-deficient HCC mice, we observed reduced levels of phospho-STAT1, autophagy, and cleaved caspase3. These responses were activated in the liver by treatment with a cyclic dinucleotide (CDN) STING agonist. Importantly, CDN treatment of mice after HCC development efficiently reduced tumor size. Initiation of CDN treatment at an even later stage of disease to allow HCC detection by MR scanning revealed that the majority of tumors regressed in response to CDN, but new tumors were also detected, which were unresponsive to CDN treatment. Overall, the modulation of the STING pathway affects the development of HCC, and holds promise for a use as a treatment of this disease, most likely in combination with other immunomodulatory treatments such as PD1 inhibitors or with standard of care.