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SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.

Macroautophagy (hereafter referred to as autophagy) is a homeostatic process that preserves cellular integrity. In mice, autophagy regulates pancreatic ductal adenocarcinoma (PDAC) development in a manner dependent on the status of the tumor suppressor gene Trp53. Studies published so far have investigated the impact of autophagy blockage in tumors arising from Trp53-hemizygous or -homozygous tissue. In contrast, in human PDACs the tumor suppressor gene TP53 is mutated rather than allelically lost, and TP53 mutants retain pathobiological functions that differ from complete allelic loss. In order to better represent the patient situation, we have investigated PDAC development in a well-characterized genetically engineered mouse model (GEMM) of PDAC with mutant Trp53 (Trp53R172H) and deletion of the essential autophagy gene Atg7. Autophagy blockage reduced PDAC incidence but had no impact on survival time in the subset of animals that formed a tumor. In the absence of Atg7, non-tumor-bearing mice reached a similar age as animals with malignant disease. However, the architecture of autophagy-deficient, tumor-free pancreata was effaced, normal acinar tissue was largely replaced with low-grade pancreatic intraepithelial neoplasias (PanINs) and insulin expressing islet β-cells were reduced. Our data add further complexity to the interplay between Atg7 inhibition and Trp53 status in tumorigenesis.

Oncogene activation and loss of tumor suppressor function changes the metabolic activity of cancer cells to drive unrestricted proliferation. Moreover, cancer cells adapt their metabolism to sustain growth and survival when access to oxygen and nutrients is restricted, such as in poorly vascularized tumor areas. We show here that p53-deficient colon cancer cells exposed to tumor-like metabolic stress in spheroid culture activated the mevalonate pathway to promote the synthesis of ubiquinone. This was essential to maintain mitochondrial electron transport for respiration and pyrimidine synthesis in metabolically compromised environments. Induction of mevalonate pathway enzyme expression in the absence of p53 was mediated by accumulation and stabilization of mature SREBP2. Mevalonate pathway inhibition by statins blocked pyrimidine nucleotide biosynthesis and induced oxidative stress and apoptosis in p53-deficient cancer cells in spheroid culture. Moreover, ubiquinone produced by the mevalonate pathway was essential for the growth of p53-deficient tumor organoids. In contrast, inhibition of intestinal hyperproliferation by statins in an Apc/KrasG12D-mutant mouse model was independent of de novo pyrimidine synthesis. Our results highlight the importance of the mevalonate pathway for maintaining mitochondrial electron transfer and biosynthetic activity in cancer cells exposed to metabolic stress. They also demonstrate that the metabolic output of this pathway depends on both genetic and environmental context. Significance: These findings suggest that p53-deficient cancer cells activate the mevalonate pathway via SREBP2 and promote the synthesis of ubiquinone that plays an essential role in reducing oxidative stress and supports the synthesis of pyrimidine nucleotide.

The notion that macroautophagy/autophagy is a potentially attractive therapeutic target for a variety of diseases, including cancer, largely stems from pre-clinical mouse studies. Most of these examine the effects of irreversible and organ confined autophagy deletion using site specific Cre-loxP recombination of the essential autophagy regulating genes Atg7 or Atg5. Model systems with the ability to impair autophagy systemically and reversibly at all disease stages would allow a more realistic approach to evaluate the consequences of authophagy inhibition as a therapeutic concept and its potential side effects. Here, we present shRNA transgenic mice that via doxycycline (DOX) regulable expression of a highly efficient miR30-E-based shRNA enabled knockdown of Atg7 simultaneously in the majority of organs, with the brain and spleen being noteable exceptions. Induced animals deteriorated rapidly and experienced profound destruction of the exocrine pancreas, severe hypoglycemia and depletion of hepatic glycogen storages. Cessation of DOX application restored apparent health, glucose homeostasis and pancreatic integrity. In a similar Atg5 knockdown model we neither observed loss of pancreatic integrity nor diminished survival after DOX treatment, but identified histological changes consistent with steatohepatitis and hepatic fibrosis in the recovery period after termination of DOX. Regulable Atg7-shRNA mice are valuable tools that will enable further studies on the role of autophagy impairment at various disease stages and thereby help to evaluate the consequences of acute autophagy inhibition as a therapeutic concept. Abbreviations: ACTB: actin, beta; AMY: amylase complex; ATG4B: autophagy related 4B, cysteine peptidase; ATG5: autophagy related 5; ATG7: autophagy related 7; Cag: CMV early enhancer/chicken ACTB promoter; Col1a1: collagen, type I, alpha 1; Cre: cre recombinase; DOX: doxycycline; GCG: glucagon; GFP: green fluorescent protein; INS: insulin; LC3: microtubule-associated protein 1 light chain 3; miR30-E: optimized microRNA backbone; NAFLD: non-alcoholic fatty liver disease; NASH: non-alcoholic steatohepatitis; PNLIP: pancreatic lipase; rtTA: reverse tetracycline transactivator protein; SQSTM1/p62: sequestome 1; TRE: tetracycline responsive element.

Lipoblastoma is a rare benign mesenchymal neoplasm that typically occurs in infancy but may also occur in older age groups and various locations. Thus, there are often numerous clinical differential diagnoses. Moreover, lipoblastomas can show a broad histologic spectrum, which can hamper the correct diagnosis, particularly in small biopsies. At the genomic level, lipoblastomas are characterized by chromosomal fusions involving the PLAG1 gene. We investigated 11 lipoblastoma samples from 10 pediatric patients (age range five months to 12 years), including one patient with local recurrence, in view of their histopathological features, and performed targeted RNA sequencing. We found a broad histological spectrum with some tumors with prominent myxoid changes, but also tumors composed mainly of mature adipocytic cells, and classified the cases according to the literature as classic (mixed), maturing, or myxoid subtype. By targeted RNA sequencing analysis, we identified characteristic PLAG1 rearrangements in 70% of the investigated cases. Moreover, these analyses revealed three novel gene fusions, two affecting the PLAG1 gene and one involving HMGA2. Besides, we performed PLAG1 immunohistochemistry and identified positive cells, typically immature adipocytic cells and spindle cells, at various numbers in all cases. However, in the maturing areas, only very sparsely positive cells were found, limiting the value of the PLAG1 immunohistochemistry as an adjunct in the diagnosis of lipoblastoma, particularly for the maturing subtype and small biopsies. The presented case series confirms the broad morphological spectrum of lipoblastoma described in the literature and underlines the value of modern molecular diagnostic approaches as a supportive diagnostic tool in challenging cases and for gaining further insights into the molecular basis of this rare mesenchymal tumor.

Deregulated expression of the MYC oncoprotein enables tumor cells to evade immune surveillance, but the mechanisms underlying this surveillance are poorly understood. We show here that endogenous MYC protects pancreatic ductal adenocarcinoma (PDAC) driven by KRASG12D and TP53R172H from eradication by the immune system. Deletion of TANK-binding kinase 1 (TBK1) bypassed the requirement for high MYC expression. TBK1 was active due to the accumulation of double-stranded RNA (dsRNA), which was derived from inverted repetitive elements localized in introns of nuclear genes. Nuclear-derived dsRNA is packaged into extracellular vesicles and subsequently recognized by toll-like receptor 3 (TLR3) to activate TBK1 and downstream MHC class I expression in an autocrine or paracrine manner before being degraded in lysosomes. MYC suppressed loading of dsRNA onto TLR3 and its subsequent degradation via association with MIZ1. Collectively, these findings suggest that MYC and MIZ1 suppress a surveillance pathway that signals perturbances in mRNA processing to the immune system, which facilitates immune evasion in PDAC. Significance: This study identifies a TBK1-dependent pathway that links dsRNA metabolism to antitumor immunity and shows that suppression of TBK1 is a critical function of MYC in pancreatic ductal adenocarcinoma.

Macroautophagy (autophagy hereafter) may promote survival and growth of spontaneous tumors, including melanoma. We utilized a genetically engineered mouse model of melanoma driven by oncogenic BrafV600E and deficiency in the Pten tumor suppressor gene in melanocytes to test the functional consequences of loss of the essential autophagy gene autophagy-related-7, Atg7. Atg7 deficiency prevented melanoma development by BrafV600E and allelic Pten loss, indicating that autophagy is essential for melanomagenesis. Moreover, BrafV600E-mutant, Pten-null, Atg7-deficient melanomas displayed accumulation of autophagy substrates and growth defects, which extended animal survival. Atg7-deleted tumors showed increased oxidative stress and senescence, a known barrier to melanomagenesis. Treatment with the BRAF inhibitor dabrafenib decreased tumor growth and induced senescence that was more pronounced in tumors with Atg7 deficiency. Thus, Atg7 promotes melanoma by limiting oxidative stress and overcoming senescence, and autophagy inhibition may be of therapeutic value by augmenting the antitumor activity of BRAF inhibitors.The essential autophagy gene Atg7 promotes development of BrafV600E-mutant, Pten-null melanomas by overcoming senescence, and deleting Atg7 facilitated senescence induction and antitumor activity of BRAF inhibition. This suggests that combinatorial BRAFV600E and autophagy inhibition may improve therapeutic outcomes in patients whose tumors have BRAFV600E/K mutations, an approach currently being explored in clinical trials.

Metabolic reprogramming is a prominent feature of clear cell renal cell carcinoma (ccRCC). Here we investigated metabolic dependencies in a panel of ccRCC cell lines using nutrient depletion, functional RNAi screening and inhibitor treatment. We found that ccRCC cells are highly sensitive to the depletion of glutamine or cystine, two amino acids required for glutathione (GSH) synthesis. Moreover, silencing of enzymes of the GSH biosynthesis pathway or glutathione peroxidases, which depend on GSH for the removal of cellular hydroperoxides, selectively reduced viability of ccRCC cells but did not affect the growth of non-malignant renal epithelial cells. Inhibition of GSH synthesis triggered ferroptosis, an iron-dependent form of cell death associated with enhanced lipid peroxidation. VHL is a major tumour suppressor in ccRCC and loss of VHL leads to stabilisation of hypoxia inducible factors HIF-1α and HIF-2α. Restoration of functional VHL via exogenous expression of pVHL reverted ccRCC cells to an oxidative metabolism and rendered them insensitive to the induction of ferroptosis. VHL reconstituted cells also exhibited reduced lipid storage and higher expression of genes associated with oxidiative phosphorylation and fatty acid metabolism. Importantly, inhibition of β-oxidation or mitochondrial ATP-synthesis restored ferroptosis sensitivity in VHL reconstituted cells. We also found that inhibition of GSH synthesis blocked tumour growth in a MYC-dependent mouse model of renal cancer. Together, our data suggest that reduced fatty acid metabolism due to inhibition of β-oxidation renders renal cancer cells highly dependent on the GSH/GPX pathway to prevent lipid peroxidation and ferroptotic cell death.

(Macro-)autophagy is an evolutionary conserved 'self-digestion program' that serves to maintain cellular metabolism and is implicated in many pathological processes such as cancer. In recent years, an increasing number of studies in murine cancer models have provided a plethora of sometimes conflicting results about the role of autophagy in cancer biology. This review summarizes these studies and raises awareness that there are situations in which autophagy blockage might indeed reduce tumor growth, but that sometimes the exact opposite is the case. It is therefore vital to mimic patient conditions in preclinical mouse experiments as thoroughly as possible before commencing clinical trials.

(Macro)autophagy delivers cellular constituents to lysosomes for degradation. Although a cytoplasmic process, autophagy-deficient cells accumulate genomic damage, but an explanation for this effect is currently unclear. We report here that inhibition of autophagy causes elevated proteasomal activity leading to enhanced degradation of checkpoint kinase 1 (Chk1), a pivotal factor for the error-free DNA repair process, homologous recombination (HR). We show that loss of autophagy critically impairs HR and that autophagy-deficient cells accrue micronuclei and sub-G1 DNA, indicators of diminished genomic integrity. Moreover, due to impaired HR, autophagy-deficient cells are hyperdependent on nonhomologous end joining (NHEJ) for repair of DNA double-strand breaks. Consequently, inhibition of NHEJ following DNA damage in the absence of autophagy results in persistence of genomic lesions and rapid cell death. Because autophagy deficiency occurs in several diseases, these findings constitute an important link between autophagy and DNA repair and highlight a synthetic lethal strategy to kill autophagy-deficient cells.

No abstract available.

Macroautophagy (hereafter referred to as autophagy) is a process in which organelles termed autophagosomes deliver cytoplasmic constituents to lysosomes for degradation. Autophagy has a major role in cellular homeostasis and has been implicated in various forms of human disease. The role of autophagy in cancer seems to be complex, with reports indicating both pro-tumorigenic and tumour-suppressive roles. Here we show, in a humanized genetically-modified mouse model of pancreatic ductal adenocarcinoma (PDAC), that autophagy's role in tumour development is intrinsically connected to the status of the tumour suppressor p53. Mice with pancreases containing an activated oncogenic allele of Kras (also called Ki-Ras)--the most common mutational event in PDAC--develop a small number of pre-cancerous lesions that stochastically develop into PDAC over time. However, mice also lacking the essential autophagy genes Atg5 or Atg7 accumulate low-grade, pre-malignant pancreatic intraepithelial neoplasia lesions, but progression to high-grade pancreatic intraepithelial neoplasias and PDAC is blocked. In marked contrast, in mice containing oncogenic Kras and lacking p53, loss of autophagy no longer blocks tumour progression, but actually accelerates tumour onset, with metabolic analysis revealing enhanced glucose uptake and enrichment of anabolic pathways, which can fuel tumour growth. These findings provide considerable insight into the role of autophagy in cancer and have important implications for autophagy inhibition in cancer therapy. In this regard, we also show that treatment of mice with the autophagy inhibitor hydroxychloroquine, which is currently being used in several clinical trials, significantly accelerates tumour formation in mice containing oncogenic Kras but lacking p53.

Autophagy is an evolutionarily conserved catabolic process that involves the entrapment of cytoplasmic components within characteristic vesicles for their delivery to and degradation within lysosomes. Alterations in autophagic signaling are found in several human diseases including cancer. Here, we describe a validated immunohistochemical protocol for the detection of LC3 puncta in human formalin-fixed, paraffin-embedded cancer specimens that can also be applied to mouse tissues. In response to systemic chemotherapy, autophagy-competent mouse tumors exhibited LC3 puncta, which did not appear in mouse cancers that had been rendered autophagy-deficient by the knockdown of Atg5 or Atg7. As compared with normal tissues, LC3 staining was moderately to highly elevated in the large majority of human cancers studied, albeit tumors of the same histological type tended to be highly heterogeneous in the number and intensity of LC3 puncta per cell. Moreover, tumor-infiltrating immune cells often were highly positive for LC3. Altogether, this protocol for LC3 staining appears suitable for the specific detection of LC3 puncta in human specimens, including tissue microarrays. We surmise that this technique can be employed for retrospective or prospective studies involving large series of human tumor samples.

(Macro)Autophagy is a phylogenetically conserved membrane-trafficking process that functions to deliver cytoplasmic cargoes to lysosomes for digestion. The process is a major mechanism for turnover of cellular constituents and is therefore critical for maintaining cellular homeostasis. Macroautophagy is characteristically distinct from other forms of autophagy due to the formation of double-membraned vesicles termed autophagosomes which encapsulate cargoes prior to fusion with lysosomes. Autophagosomes contain an integral membrane-bound form (LC3-II) of the microtubule-associated protein 1 light chain 3 β (MAP1LC3B), which has become a gold-standard marker to detect accumulation of autophagosomes and thereby changes in macroautophagy. Due to the role played by macroautophagy in various diseases, the detection of autophagosomes in tissue sections is frequently desired. To date, however, the detection of endogenous LC3-II on paraffin-embedded tissue sections has proved problematic. We report here a simple, optimized and validated method for the detection of LC3-II by immunohistochemistry in human and mouse tissue samples that we believe will be a useful resource for those wishing to study macroautophagy ex vivo.

Autophagy is an evolutionarily conserved, catabolic process that involves the entrapment of cytoplasmic components within characteristic vesicles for their delivery to and degradation within lysosomes. Autophagy is regulated via a group of genes called AuTophaGy-related genes and is executed at basal levels in virtually all cells as a homeostatic mechanism for maintaining cellular integrity. The levels and cargos of autophagy can be modulated in response to a variety of intra- and extracellular cues to bring about specific and selective events. Autophagy is a multifaceted process and alterations in autophagic signalling pathways are frequently found in cancer and many other diseases. During tumour development and in cancer therapy, autophagy has paradoxically been reported to have roles in promoting both cell survival and cell death. In addition, autophagy has been reported to control other processes relevant to the aetiology of malignant disease, including oxidative stress, inflammation and both innate and acquired immunity. It is the aim of this review to describe the molecular basis and the signalling events that control autophagy in mammalian cells and to summarize the cellular functions that contribute to tumourigenesis when autophagy is perturbed.

Autophagy is a catabolic membrane-trafficking process that leads to sequestration and degradation of intracellular material within lysosomes. It is executed at basal levels in every cell and promotes cellular homeostasis by regulating organelle and protein turnover. In response to various forms of cellular stress, however, the levels and cargoes of autophagy can be modulated. In nutrient-deprived states, for example, autophagy can be activated to degrade cargoes for cell-autonomous energy production to promote cell survival. In other contexts, in contrast, autophagy has been shown to contribute to cell death. Given these dual effects in regulating cell viability, it is no surprise that autophagy has implications in both the genesis and treatment of malignant disease. In this review, we provide a comprehensive appraisal of the way in which oncogenes and tumour suppressor genes regulate autophagy. In addition, we address the current evidence from human cancer and animal models that has aided our understanding of the role of autophagy in tumour progression. Finally, the potential for targeting autophagy therapeutically is discussed in light of the functions of autophagy at different stages of tumour progression and in normal tissues.

Introduction: Besides triggering effector cascades of the immune system, CD20-directed immunotherapy induces programmed death of malignant B-cells. Therapy-mediated senescence is a long-term growth arrest of metabolically active tumor cells with prognostic relevance in preclinical lymphoma models, especially under conditions with impaired integrity of apoptotic pathways. Several therapeutic antibodies have been implied to modulate senescence levels in human tumor cells. However, the contribution of senescence to outcome to rituximab-based immunochemotherapy in B-cell lymphoma remains to be elucidated. Methods: We applied CD20-directed immunotherapy or the anti-lymphoma drug adriamycin (ADR, a well-known inducer of cellular senescence) to B-cell lines and primary EBV-transformed B-lymphoblastoid cells. Growth characteristics, senescence-associated β-galactosidase (SA-β-Gal) reactivity, selected components of the senescence-associated secretory phenotype (SASP) as well as intracellular reactive oxygen species (ROS) were analyzed. Senescence-related gene sets were probed for enrichment in a publically available sequential data set derived from patients with chronic lymphocytic B-cell leukemia (CLL) undergoing their first cycle of rituximab-based immunochemotherapy. Results: Major features of cellular senescence could be detected in response to CD20-directed immunotherapy as well as ADR in B-cell lines and primary EBV-transformed B-lymphoblastoid cells in vitro. Moreover, additional rituximab-based treatment increased levels of SA-β-Gal-reactive cells as well as secretion of the SASP components interleukin-6 and 8 and further reduced cell fractions in S-phase incorporating 5-bromodeoxyuridine when compared to application of ADR as a single agent. Elevated ROS levels were detected as a response to CD20-directed therapy and rituximab-mediated senescence was entirely abrogated upon prior administration of the ROS scavenger N-acetylcysteine. Moreover, we found senescent-related gene sets that had been inferred from relevant publications or were defined based on our own Chip-on-Chip analyses in human fibroblasts undergoing oncogene-induced senescence to be selectively enriched for in post-treatment samples from CLL patients later achieving partial or complete remission, but not with stable or progressive disease. Discussion: Our results show ROS-mediated lymphoma cell senescence as a hitherto unrecognized consequence of rituximab-based immunotherapy and argue for a contribution of enhanced senescence levels after combined immunochemotherapy to the clinical superiority of treatment regimens that include rituximab. Moreover, sequential gene set enrichment analyses hint towards an association of therapy-induced senescence and favorable outcome to immunochemotherapy in CLL. Our data warrant further investigation of cellular senescence as an effector program of antineoplastic treatment in human lymphoma. Disclosures No relevant conflicts of interest to declare.

Abstract 1827 Introduction: Targeting the B-cell antigen CD20 in addition to conventional DNA damaging chemotherapy has dramatically improved patient prognosis in a variety of B-cell malignancies without overtly increasing treatment toxicity. The therapeutic benefit of rituximab as the prototypic anti-CD20 compound has often been attributed to classical antibody effector functions of the immune system, and, to some extent, to induction of apoptosis as well. Interestingly, there is no consensus regarding the intracellular pathways triggered by CD20 signaling. Cellular senescence, a terminal growth arrest condition, complements apoptosis as another cell-cycle exit program that is acutely inducible in malignant cells in response to DNA damaging chemotherapy, and is associated with favorable treatment outcome in preclinical cancer models. Senescent cells are characterized by retained viability, distinct morphology, and a cytokine secretion pattern that mediates an intense cross-talk to the microenvironment and cells of the immune system. Results: Here, we provide evidence for induction of cellular senescence by rituximab in cell lines derived from aggressive and indolent human B-cell lymphomas. Mimicking the in vivo presence of Fc-receptor-bearing cells by addition of a F(ab’)2-fragment directed against the constant part of human IgG, we observed diminished growth, altered cellular morphology and senescence-associated ß-galactosidase reactivity after anti-CD20 immunotherapy in vitro. Strikingly, when applied together with a moderate concentration of the anthracycline adriamycin, anti-CD20 treatment strongly augmented the fraction of lymphoma cells entering a senescent cell-cycle arrest. Expression and secretion of cytokines typically associated with cellular senescence (such as IL-6 and IL-8) were increased afterin vitro immunochemotherapy (analysed by RQ-PCR and fluorescent bead immunoassays of media supernatants). Unexpectedly, we detected higher levels of DNA damage signaling markers (phosphorylated H2AX [serine 139] and phosphorylated p53 [serine 15]) with anti-CD20 co-treatment compared to single agent adriamycin application. Discussion: Our data demonstrate the induction of cellular senescence, which has a profound impact on tumor biology and tumor/host interactions in preclinical tumor models, as a consequence of standard lymphoma immunotherapy in humans. The anti-CD20-mediated augmentation of the anthracycline-induced DNA damage response provides a mechanistic explanation for the stronger induction of the senescent phenotype after immunochemotherapy and may contribute to the clinical superiority of treatment regimens that include rituximab. Further elucidation of the synergy between antineoplastic agents and CD20-directed compounds with respect to induction of cellular senescence as a favorable treatment outcome opens the clinical perspective of specifically engaging the senescence program by therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53 fl/fl :lsl-KRas G12D/wt . Developing tumors were indistinguishable from Trp53 fl/fl :lsl-KRas G12D/wt -derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research.