Pathologists' perspective on the study design, analysis, and interpretation of proliferative lesions in a lifetime rodent carcinogenicity bioassay of sucralose.

Sucralose, a sugar substitute first approved for use in 1991, is a non-caloric sweetener regulated globally as a food additive. Based on numerous experimental animal studies (dating to the 1980s) and human epidemiology studies, international health agencies have determined that sucralose is safe when consumed as intended. A single lifetime rodent carcinogenicity bioassay conducted by the Ramazzini Institute (RI) reported that mice fed diets containing sucralose develop hematopoietic neoplasia, but controversy continues regarding the validity and relevance of these data for predicting health effects in humans. The present paper addresses the controversy by providing the perspective of experienced pathologists on sucralose-related animal toxicity and carcinogenicity data generally, and the RI carcinogenicity bioassay findings specifically, using results from publicly available papers and international regulatory authority decisions. In the authors' view, flaws in the design, methodology, data evaluation, and reporting of the RI carcinogenicity bioassay for sucralose diminish the value of the data as evidence that this agent represents a carcinogenic hazard to humans. This limitation will remain until the RI bioassay is repeated under Good Laboratory Practices and the design, data, and accuracy of the pathology diagnoses and interpretations are reviewed by qualified pathologists with experience in evaluating potential chemically-induced carcinogenic hazards.

PAX3-FOXO1 dictates myogenic reprogramming and rhabdomyosarcoma identity in endothelial progenitors.

Fusion-positive rhabdomyosarcoma (FP-RMS) driven by the expression of the PAX3-FOXO1 (P3F) fusion oncoprotein is an aggressive subtype of pediatric rhabdomyosarcoma. FP-RMS histologically resembles developing muscle yet occurs throughout the body in areas devoid of skeletal muscle highlighting that FP-RMS is not derived from an exclusively myogenic cell of origin. Here we demonstrate that P3F reprograms mouse and human endothelial progenitors to FP-RMS. We show that P3F expression in aP2-Cre expressing cells reprograms endothelial progenitors to functional myogenic stem cells capable of regenerating injured muscle fibers. Further, we describe a FP-RMS mouse model driven by P3F expression and Cdkn2a loss in endothelial cells. Additionally, we show that P3F expression in TP53-null human iPSCs blocks endothelial-directed differentiation and guides cells to become myogenic cells that form FP-RMS tumors in immunocompromised mice. Together these findings demonstrate that FP-RMS can originate from aberrant development of non-myogenic cells driven by P3F.

Genome-wide mapping of cancer dependency genes and genetic modifiers of chemotherapy in high-risk hepatoblastoma.

A lack of relevant genetic models and cell lines hampers our understanding of hepatoblastoma pathogenesis and the development of new therapies for this neoplasm. Here, we report an improved MYC-driven hepatoblastoma-like murine model that recapitulates the pathological features of embryonal type of hepatoblastoma, with transcriptomics resembling the high-risk gene signatures of the human disease. Single-cell RNA-sequencing and spatial transcriptomics identify distinct subpopulations of hepatoblastoma cells. After deriving cell lines from the mouse model, we map cancer dependency genes using CRISPR-Cas9 screening and identify druggable targets shared with human hepatoblastoma (e.g., CDK7, CDK9, PRMT1, PRMT5). Our screen also reveals oncogenes and tumor suppressor genes in hepatoblastoma that engage multiple, druggable cancer signaling pathways. Chemotherapy is critical for human hepatoblastoma treatment. A genetic mapping of doxorubicin response by CRISPR-Cas9 screening identifies modifiers whose loss-of-function synergizes with (e.g., PRKDC) or antagonizes (e.g., apoptosis genes) the effect of chemotherapy. The combination of PRKDC inhibition and doxorubicin-based chemotherapy greatly enhances therapeutic efficacy. These studies provide a set of resources including disease models suitable for identifying and validating potential therapeutic targets in human high-risk hepatoblastoma.

The many faces of mouse histiocytic sarcoma in C57BL/6J mice.

Histiocytic sarcoma is a tumor of the hematopoietic system considered to be derived from macrophages. Although rare in humans, it occurs frequently in mice. Histiocytic sarcoma can be a difficult tumor to diagnose due to its diverse cellular morphologies, growth patterns, and organ distributions. The varying morphology of histiocytic sarcomas makes it easy to confuse them with other types of neoplasia, including hepatic hemangiosarcoma, uterine schwannoma, leiomyosarcoma, uterine stromal cell tumor, intramedullary osteosarcoma, and myeloid leukemia. As such, immunohistochemistry (IHC) is often needed to differentiate histiocytic sarcomas from other common tumors in mice that they can morphologically mimic. The goal of this article is to present a broader perspective of the diverse cellular morphologies, growth patterns, organ distributions, and IHC labeling of histiocytic sarcomas encountered by the authors. This article describes these features in a set of 62 mouse histiocytic sarcomas, including the IHC characterization of the tumors using a panel of markers for the macrophage antigens F4/80, IBA1, MAC2, CD163, CD68, and lysozyme, and describes differentiating features of histiocytic sarcomas from other morphologically similar tumors. The genetic changes underlying the pathogenesis of histiocytic sarcoma in humans are beginning to be elucidated, but this is difficult due to its rarity. The higher prevalence of this tumor in mice provides opportunities to investigate mechanisms of its development and to test potential treatments.

ATM inhibition enhances the efficacy of radiation across distinct molecular subgroups of pediatric high-grade glioma.

BACKGROUND: Pediatric high-grade glioma (pHGG) is largely incurable and accounts for most brain tumor-related deaths in children. Radiation is a standard therapy, yet the benefit from this treatment modality is transient, and most children succumb to disease within 2 years. Recent large-scale genomic studies suggest that pHGG has alterations in DNA damage response (DDR) pathways that induce resistance to DNA damaging agents. The aim of this study was to evaluate the therapeutic potential and molecular consequences of combining radiation with selective DDR inhibition in pHGG. METHODS: We conducted an unbiased screen in pHGG cells that combined radiation with clinical candidates targeting the DDR and identified the ATM inhibitor AZD1390. Subsequently, we profiled AZD1390 + radiation in an extensive panel of early passage pHGG cell lines, mechanistically characterized response to the combination in vitro in sensitive and resistant cells and evaluated the combination in vivo using TP53 wild-type and TP53 mutant orthotopic xenografts. RESULTS: AZD1390 significantly potentiated radiation across molecular subgroups of pHGG by increasing mutagenic nonhomologous end joining and augmenting genomic instability. In contrast to previous reports, ATM inhibition significantly improved the efficacy of radiation in both TP53 wild-type and TP53 mutant isogenic cell lines and distinct orthotopic xenograft models. Furthermore, we identified a novel mechanism of resistance to AZD1390 + radiation that was marked by an attenuated ATM pathway response which dampened sensitivity to ATM inhibition and induced synthetic lethality with ATR inhibition. CONCLUSIONS: Our study supports the clinical evaluation of AZD1390 in combination with radiation in pediatric patients with HGG.

Pathologists' perspective on the study design, analysis, and interpretation of proliferative lesions in lifetime and prenatal rodent carcinogenicity bioassays of aspartame.

Aspartame, an artificial sweetener commonly used as a sugar substitute, is currently authorized for use in more than 100 countries. Hundreds of studies, conducted in various countries dating back to the 1970s, have shown that aspartame is safe at real-world exposure levels. Furthermore, multiple human epidemiology studies have provided no indication that consumption of aspartame induces cancer. Given the continued controversy surrounding the Ramazzini Institute's (RI) studies suggesting that aspartame is a carcinogenic hazard in rodents and evaluation by the International Agency for Research on Cancer, this report aims to provide the perspective of experienced pathologists on publicly available pathology data regarding purported proliferative lesions in liver, lung, lymphoid organs, and mammary gland as well as their implications for human risk assessment as reported for three lifetime rodent carcinogenicity bioassays of aspartame conducted at the RI. In the authors' view, flaws in the design, methodology and reporting of the RI aspartame studies limit the utility of the data sets as evidence that this agent represents a carcinogenic hazard. Therefore, all three RI studies, and particularly the accuracy of their pathology diagnoses and interpretations, should be rigorously reviewed by qualified and experienced veterinary toxicologic pathologists in assessing aspartame's carcinogenic risk.

Genetic context of oncogenic drivers dictates vascular sarcoma development in aP2-Cre mice.

Angiosarcomas are aggressive vascular sarcomas that arise from endothelial cells and have an extremely poor prognosis. Because of the rarity of angiosarcomas, knowledge of molecular drivers and optimized treatment strategies is lacking, highlighting the need for in vivo models to study the disease. Previously, we generated genetically engineered mouse models of angiosarcoma driven by aP2-Cre-mediated biallelic loss of Dicer1 or conditional activation of KrasG12D with Cdkn2a loss that histologically and genetically resemble human tumors. In the present study, we found that DICER1 functions as a potent tumor suppressor and its deletion, in combination with either KRASG12D expression or Cdkn2a loss, is associated with angiosarcoma development. Independent of the genetic driver, the mTOR pathway was activated in all murine angiosarcoma models. Direct activation of the mTOR pathway by conditional deletion of Tsc1 with aP2-Cre resulted in tumors that resemble intermediate grade human kaposiform hemangioendotheliomas, indicating that mTOR activation was not sufficient to drive the malignant angiosarcoma phenotype. Genetic dissection of the spectrum of vascular tumors identified genes specifically regulated in the aggressive murine angiosarcomas that are also enriched in human angiosarcoma. The genetic dissection driving the transition across the malignant spectrum of endothelial sarcomas provides an opportunity to identify key determinants of the malignant phenotype, novel therapies for angiosarcoma, and novel in vivo models to further explore angiosarcoma pathogenesis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Synthetic essentiality between PTEN and core dependency factor PAX7 dictates rhabdomyosarcoma identity.

PTEN promoter hypermethylation is nearly universal and PTEN copy number loss occurs in ~25% of fusion-negative rhabdomyosarcoma (FN-RMS). Here we show Pten deletion in a mouse model of FN-RMS results in less differentiated tumors more closely resembling human embryonal RMS. PTEN loss activated the PI3K pathway but did not increase mTOR activity. In wild-type tumors, PTEN was expressed in the nucleus suggesting loss of nuclear PTEN functions could account for these phenotypes. Pten deleted tumors had increased expression of transcription factors important in neural and skeletal muscle development including Dbx1 and Pax7. Pax7 deletion completely rescued the effects of Pten loss. Strikingly, these Pten;Pax7 deleted tumors were no longer FN-RMS but displayed smooth muscle differentiation similar to leiomyosarcoma. These data highlight how Pten loss in FN-RMS is connected to a PAX7 lineage-specific transcriptional output that creates a dependency or synthetic essentiality on the transcription factor PAX7 to maintain tumor identity.

The Common Germline TP53-R337H Mutation Is Hypomorphic and Confers Incomplete Penetrance and Late Tumor Onset in a Mouse Model.

The TP53-R337H founder mutation exists at a high frequency throughout southern Brazil and represents one of the most common germline TP53 mutations reported to date. It was identified in pediatric adrenocortical tumors in families with a low incidence of cancer. The R337H mutation has since been found in association with early-onset breast cancers and Li-Fraumeni syndrome (LFS). To study this variability in tumor susceptibility, we generated a knockin mutant p53 mouse model (R334H). Endogenous murine p53-R334H protein was naturally expressed at high levels in multiple tissues and was functionally compromised in a tissue- and stress-specific manner. Mutant p53-R334H mice developed tumors with long latency and incomplete penetrance, consistent with many human carriers being at a low but elevated risk for cancer. These findings suggest the involvement of additional cooperating genetic alterations when TP53-R337H occurs in the context of LFS, which has important implications for genetic counseling and long-term clinical follow-up. SIGNIFICANCE: A p53-R334H knockin mouse serves as an important model for studying the most common inherited germline TP53 mutation (R337H) that is associated with variable tumor susceptibility.

Development of Mast Cell and Eosinophil Hyperplasia and HLH/MAS-Like Disease in NSG-SGM3 Mice Receiving Human CD34+ Hematopoietic Stem Cells or Patient-Derived Leukemia Xenografts.

Immunocompromised mouse strains expressing human transgenes are being increasingly used in biomedical research. The genetic modifications in these mice cause various cellular responses, resulting in histologic features unique to each strain. The NSG-SGM3 mouse strain is similar to the commonly used NSG (NOD scid gamma) strain but expresses human transgenes encoding stem cell factor (also known as KIT ligand), granulocyte-macrophage colony-stimulating factor, and interleukin 3. This report describes 3 histopathologic features seen in these mice when they are unmanipulated or after transplantation with human CD34+ hematopoietic stem cells (HSCs), virally transduced hCD34+ HSCs, or a leukemia patient-derived xenograft. The first feature is mast cell hyperplasia: unmanipulated, naïve mice develop periductular pancreatic aggregates of murine mast cells, whereas mice given the aforementioned human cells develop a proliferative infiltrative interstitial pancreatic mast cell hyperplasia but with human mast cells. The second feature is the predisposition of NSG-SGM3 mice given these human cells to develop eosinophil hyperplasia. The third feature, secondary hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS)-like disease, is the most pronounced in both its clinical and histopathologic presentations. As part of this disease, a small number of mice also have histiocytic infiltration of the brain and spinal cord with subsequent neurologic or vestibular signs. The presence of any of these features can confound accurate histopathologic interpretation; therefore, it is important to recognize them as strain characteristics and to differentiate them from what may be experimentally induced in the model being studied.

Spectrum of Posttransplant Lymphoproliferations in NSG Mice and Their Association With EBV Infection After Engraftment of Pediatric Solid Tumors.

Pediatric patients receiving solid organ transplants may develop lymphoproliferative diseases, including graft-versus-host disease (GvHD) and posttransplant lymphoproliferative diseases (PTLDs). We characterized lesions in 11 clinically ill NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice that received pediatric-patient-derived solid tumors (PDXs) and developed immunodeficiency-associated lymphoproliferations comparable to GvHD and PTLDs over a period of 46 to 283 days after implantation. Lymphoproliferations were diffusely positive for human-specific biomarkers, including NUMA1, CD45, and CD43, but lacked immunoreactivity for murine CD45. Human immune cells were CD3-positive, with subsets having immunoreactivity for CD4 and CD8 as well as PAX5, CD79a, and IRF4, resulting from populations of human T and B cells present within the xenotransplants. Tissues and organs infiltrated included mucocutaneous zones (oral cavity and perigenital and perianal regions), haired skin, tongue, esophagus, forestomach, thyroid, salivary glands, lungs, liver, kidneys, spleen, lymph nodes, bone marrow, and brain. In 4 of 5 mice with PTLD, Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) were detected by in situ hybridization in PAX5+ human B cells associated with the PDX (n = 1/4) or with engrafted human immune cells at other anatomic locations (n = 4/11). One of the 4 mice had an EBV-associated human large B-cell lymphoma. NSG mice receiving xenotransplants can develop combinations of GvHD, EBV-driven PTLD, and B-cell lymphoma similar to those occurring in human pediatric patients. Therefore, pediatric xenotransplants should undergo histopathologic and immunohistochemical assessment upon collection to ensure that the specimen is not a lymphoma and does not contain lymphoma cells because these neoplasms can morphologically mimic small round blue cell pediatric solid tumors.

Large 1p36 Deletions Affecting Arid1a Locus Facilitate Mycn-Driven Oncogenesis in Neuroblastoma.

Loss of heterozygosity (LOH) at 1p36 occurs in multiple cancers, including neuroblastoma (NBL). MYCN amplification and 1p36 deletions tightly correlate with markers of tumor aggressiveness in NBL. Although distal 1p36 losses associate with single-copy MYCN tumors, larger deletions correlate with MYCN amplification, indicating two tumor suppressor regions in 1p36, only one of which facilitates MYCN oncogenesis. To better define this region, we genome-edited the syntenic 1p36 locus in primary mouse neural crest cells (NCCs), a putative NBL cell of origin. In in vitro cell transformation assays, we show that Chd5 loss confers most of the MYCN-independent tumor suppressor effects of 1p36 LOH. In contrast, MYCN-driven tumorigenesis selects for NCCs with Arid1a deletions from a pool of NCCs with randomly sized 1p36 deletions, establishing Arid1a as the MYCN-associated tumor suppressor. Our findings reveal that Arid1a loss collaborates with oncogenic MYCN and better define the tumor suppressor functions of 1p36 LOH in NBL.

Morphologic and Immunohistochemical Characterization of Spontaneous Lymphoma/Leukemia in NSG Mice.

The NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ strain (NOD scid gamma, NSG) is a severely immunodeficient inbred laboratory mouse used for preclinical studies because it is amenable to engraftment with human cells. Combining scid and Il2rgnull mutations results in severe immunodeficiency by impairing the maturation, survival, and functionality of interleukin 2-dependent immune cells, including T, B, and natural killer lymphocytes. While NSG mice are reportedly resistant to developing spontaneous lymphomas/leukemias, there are reports of hematopoietic cancers developing. In this study, we characterized the immunophenotype of spontaneous lymphoma/leukemia in 12 NSG mice (20 to 38 weeks old). The mice had a combination of grossly enlarged thymus, spleen, or lymph nodes and variable histologic involvement of the bone marrow and other tissues. All 12 lymphomas were diffusely CD3, TDT, and CD4 positive, and 11 of 12 were also positive for CD8, which together was consistent with precursor T-cell lymphoblastic lymphoma/leukemia (pre-T-LBL). A subset of NSG tissues from all mice and neoplastic lymphocytes from 8 of 12 cases had strong immunoreactivity for retroviral p30 core protein, suggesting an association with a viral infection. These data highlight that NSG mice may develop T-cell lymphoma at low frequency, necessitating the recognition of this spontaneously arising disease when interpreting studies.

Forty-five patient-derived xenografts capture the clinical and biological heterogeneity of Wilms tumor.

The lack of model systems has limited the preclinical discovery and testing of therapies for Wilms tumor (WT) patients who have poor outcomes. Herein, we establish 45 heterotopic WT patient-derived xenografts (WTPDX) in CB17 scid-/- mice that capture the biological heterogeneity of Wilms tumor (WT). Among these 45 total WTPDX, 6 from patients with diffuse anaplastic tumors, 9 from patients who experienced disease relapse, and 13 from patients with bilateral disease are included. Early passage WTPDX show evidence of clonal selection, clonal evolution and enrichment of blastemal gene expression. Favorable histology WTPDX are sensitive, whereas unfavorable histology WTPDX are resistant to conventional chemotherapy with vincristine, actinomycin-D, and doxorubicin given singly or in combination. This WTPDX library is a unique scientific resource that retains the spectrum of biological heterogeneity present in WT and provides an essential tool to test targeted therapies for WT patient groups with poor outcomes.

Nonproliferative and Proliferative Lesions of the Rat and Mouse Hematolymphoid System.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative changes in rats and mice. The purpose of this publication is to provide a standardized nomenclature for classifying changes observed in the hematolymphoid organs, including the bone marrow, thymus, spleen, lymph nodes, mucosa-associated lymphoid tissues, and other lymphoid tissues (serosa-associated lymphoid clusters and tertiary lymphoid structures) with color photomicrographs illustrating examples of the lesions. Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. The nomenclature for these organs is divided into 3 terminologies: descriptive, conventional, and enhanced. Three terms are listed for each diagnosis. The rationale for this approach and guidance for its application to toxicologic pathology are described in detail below.

Immunophenotyping of Murine Precursor B-Cell Leukemia/Lymphoma: A Comparison of Immunohistochemistry and Flow Cytometry.

In humans and in mouse models, precursor B-cell lymphoblastic leukemia (B-ALL)/lymphoblastic lymphoma (B-LBL) can be classified as either the pro-B or pre-B subtype. This is based on the expression of antigens associated with the pro-B and pre-B stages of B-cell development. Antigenic markers can be detected by flow cytometry or immunohistochemistry (IHC), but no comparison of results from these techniques has been reported for murine B-ALL/LBL. In our analysis of 30 cases induced by chemical or viral mutagenesis on a WT or Pax5+/- background, 18 (60%) were diagnosed as pro-B by both flow cytometry and IHC. Discordant results were found for 12 (40%); 6 were designated pro-B by IHC and pre-B by flow cytometry and the reverse for the remaining 6 cases. Discordance occurred because different markers were used to define the pro-B-to-pre-B transition by IHC vs flow cytometry. IHC expression of cytoplasmic IgM (µIgM) defined the pre-B stage, whereas the common practice of using CD25 as a surrogate marker in flow cytometry was employed here. These results show that CD25 and µIgM are not always concurrently expressed in B-ALL/LBL, in contrast to normal B-cell development. Therefore, when subtyping B-ALL/LBL in mice, an IHC panel of B220, PAX5, TdT, c-Kit/CD117, CD43, IgM, and ΚLC should be considered. For flow cytometry, cytoplasmic IgM may be an appropriate marker in conjunction with the surface markers B220, CD19, CD43, c-Kit/CD117, BP-1, and CD25.

Hedgehog Pathway Drives Fusion-Negative Rhabdomyosarcoma Initiated From Non-myogenic Endothelial Progenitors.

Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma that histologically resembles embryonic skeletal muscle. RMS occurs throughout the body and an exclusively myogenic origin does not account for RMS occurring in sites devoid of skeletal muscle. We previously described an RMS model activating a conditional constitutively active Smoothened mutant (SmoM2) with aP2-Cre. Using genetic fate mapping, we show SmoM2 expression in Cre-expressing endothelial progenitors results in myogenic transdifferentiation and RMS. We show that endothelium and skeletal muscle within the head and neck arise from Kdr-expressing progenitors, and that hedgehog pathway activation results in aberrant expression of myogenic specification factors as a potential mechanism driving RMS genesis. These findings suggest that RMS can originate from aberrant development of non-myogenic cells.

Globule Leukocytes and Other Mast Cells in the Mouse Intestine.

Only 2 major mast cell (MC) subtypes are commonly recognized in the mouse: the large connective tissue mast cells (CTMCs) and the mucosal mast cells (MMCs). Interepithelial mucosal inflammatory cells, most commonly identified as globule leukocytes (GLs), represent a third MC subtype in mice, which we term interepithelial mucosal mast cells (ieMMCs). This term clearly distinguishes ieMMCs from lamina proprial MMCs (lpMMCs) while clearly communicating their common MC lineage. Both lpMMCs and ieMMCs are rare in normal mouse intestinal mucosa, but increased numbers of ieMMCs are seen as part of type 2 immune responses to intestinal helminth infections and in food allergies. Interestingly, we found that increased ieMMCs were consistently associated with decreased mucosal inflammation and damage, suggesting that they might have a role in controlling helminth-induced immunopathology. We also found that ieMMC hyperplasia can develop in the absence of helminth infections, for example, in Treg-deficient mice, Arf null mice, some nude mice, and certain graft-vs-host responses. Since tuft cell hyperplasia plays a critical role in type 2 immune responses to intestinal helminths, we looked for (but did not find) any direct relationship between ieMMC and tuft cell numbers in the intestinal mucosa. Much remains to be learned about the differing functions of ieMMCs and lpMMCs in the intestinal mucosa, but an essential step in deciphering their roles in mucosal immune responses will be to apply immunohistochemistry methods to consistently and accurately identify them in tissue sections.

ATM-deficiency increases genomic instability and metastatic potential in a mouse model of pancreatic cancer.

Germline mutations in ATM (encoding the DNA-damage signaling kinase, ataxia-telangiectasia-mutated) increase Familial Pancreatic Cancer (FPC) susceptibility, and ATM somatic mutations have been identified in resected human pancreatic tumors. Here we investigated how Atm contributes to pancreatic cancer by deleting this gene in a murine model of the disease expressing oncogenic Kras (KrasG12D). We show that partial or total ATM deficiency cooperates with KrasG12D to promote highly metastatic pancreatic cancer. We also reveal that ATM is activated in pancreatic precancerous lesions in the context of DNA damage and cell proliferation, and demonstrate that ATM deficiency leads to persistent DNA damage in both precancerous lesions and primary tumors. Using low passage cultures from primary tumors and liver metastases we show that ATM loss accelerates Kras-induced carcinogenesis without conferring a specific phenotype to pancreatic tumors or changing the status of the tumor suppressors p53, p16Ink4a and p19Arf. However, ATM deficiency markedly increases the proportion of chromosomal alterations in pancreatic primary tumors and liver metastases. More importantly, ATM deficiency also renders murine pancreatic tumors highly sensitive to radiation. These and other findings in our study conclusively establish that ATM activity poses a major barrier to oncogenic transformation in the pancreas via maintaining genomic stability.