Mouse lemurs are the smallest and fastest reproducing primates, as well as one of the most abundant, and they are emerging as a model organism for primate biology, behaviour, health and conservation. Although much has been learnt about their ecology and phylogeny in Madagascar and their physiology, little is known about their cellular and molecular biology. Here we used droplet-based and plate-based single-cell RNA sequencing to create Tabula Microcebus, a transcriptomic atlas of 226,000 cells from 27 mouse lemur organs opportunistically obtained from four donors clinically and histologically characterized. Using computational cell clustering, integration and expert cell annotation, we define and biologically organize more than 750 lemur molecular cell types and their full gene expression profiles. This includes cognates of most classical human cell types, including stem and progenitor cells, and differentiating cells along the developmental trajectories of spermatogenesis, haematopoiesis and other adult tissues. We also describe dozens of previously unidentified or sparsely characterized cell types. We globally compare expression profiles to define the molecular relationships of cell types across the body, and explore primate cell and gene expression evolution by comparing lemur transcriptomes to those of human, mouse and macaque. This reveals cell-type-specific patterns of primate specialization and many cell types and genes for which the mouse lemur provides a better human model than mouse 1 . The atlas provides a cellular and molecular foundation for studying this model primate and establishes a general approach for characterizing other emerging model organisms.

Telomerase is intimately associated with stem cells and cancer, because it catalytically elongates telomeres-nucleoprotein caps that protect chromosome ends(1). Overexpression of telomerase reverse transcriptase (TERT) enhances the proliferation of cells in a telomere-independent manner(2-8), but so far, loss-of-function studies have provided no evidence that TERT has a direct role in stem cell function. In many tissues, homeostasis is shaped by stem cell competition, a process in which stem cells compete on the basis of inherent fitness. Here we show that conditional deletion of Tert in the spermatogonial stem cell (SSC)-containing population in mice markedly impairs competitive clone formation. Using lineage tracing from the Tert locus, we find that TERT-expressing SSCs yield long-lived clones, but that clonal inactivation of TERT promotes stem cell differentiation and a genome-wide reduction in open chromatin. This role for TERT in competitive clone formation occurs independently of both its reverse transcriptase activity and the canonical telomerase complex. Inactivation of TERT causes reduced activity of the MYC oncogene, and transgenic expression of MYC in the TERT-deleted pool of SSCs efficiently rescues clone formation. Together, these data reveal a catalytic-activity-independent requirement for TERT in enhancing stem cell competition, uncover a genetic connection between TERT and MYC and suggest that a selective advantage for stem cells with high levels of TERT contributes to telomere elongation in the male germline during homeostasis and ageing.

Abstract Telomere length is an important biomarker of cellular replicative potential and aging, but existing measurement methods are limited in resolution and accuracy. During human carcinogenesis, telomeres shorten profoundly in the earliest stages that precede tumor invasion. Telomere shortening can either halt cancer progression by inducing senescence and cell death or can promote cancer development by destabilizing chromosomes and generating chromosomal rearrangements. Stabilization of telomeres, typically by the upregulation of telomerase, is required for progression of nearly all human cancers. Here, we deploy digital telomere measurement by nanopore sequencing to understand how distributions of human telomere length change during aging and following carcinogenesis. We measure telomere attrition and de novo elongation with unprecedented resolution in genetically defined populations of human stem cells, in cells from the peripheral blood of healthy individuals and patients with genetic defects in telomere maintenance, cancer patients, and cancer cell lines. We find that human aging is accompanied by a progressive loss of long telomeres and an accumulation of shorter telomeres in white blood cells. In patients with inherited defects in telomere maintenance, the accumulation of short telomeres is more pronounced and correlates with phenotypic severity. We apply machine learning to train a binary classification model that distinguishes healthy individuals from those with telomere biology disorders. Next, we investigated the telomere lengths of a cohort of twenty colorectal tumors with patient-matched healthy colonic epithelia and found that two-thirds of colorectal tumors have telomere length distributions significantly shorter than the surrounding normal tissue. Finally, we explore the utility of telomere length distributions in cancer cell lines as clinically actionable disease biomarkers. This sequencing and bioinformatic pipeline will advance our understanding of telomere maintenance mechanisms and the use of telomere length as a clinical biomarker of aging and disease. Citation Format: Santiago Sanchez, Jessica Gu, Anudeep Golla, William Shomali, Annika Martin, Dirk Hockemeyer, Sharon Savage, Steven Artandi. High resolution telomere measurements in human cancer and aging using long-read nanopore sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB185.

Abstract Salivary gland homeostasis and regeneration post-radiotherapy depend significantly on stem/progenitor cells. However, the lineage of SMG progenitor cells is not as well defined as in other normal organs, and better understanding of this population would be important for their future clinical application for tissue regeneration. Using a mouse strain expressing regulated CreERT2 recombinase from the endogenous Tert locus, we identify a distinct Terthigh cell population that are located in the ductal region throughout the submandibular gland (SMG), express ASCL3 (another progenitor cell marker) and can give rise to ductal cells during homeostasis. In contrast, Terthigh cells are found to repopulate both ductal and acinar cells at one year of tracing after radiotherapy. Terthigh cells from adult SMG retain self-renewal capacity when subjected in vitro culture, are resistant to radiation cell kill, and display enhanced proliferative activity post-radiation. Similarly, primary human SMG cells with high Tert expression display enhanced cell survival after radiotherapy. RNA sequencing reveals upregulation of the cell cycling and oxidative stress response pathways these cells after radiation. Mechanistically, Tert appears to modulate cell survival and ROS level in SMG spheres following radiation damage. Collectively, these data suggest that Terthigh cells constitute a novel subset of SMG progenitor cells located throughout the ductal region of the gland that can help to repopulate both ductal and acinar cells after RT damage. Citation Format: Li Guan, Vignesh Viswanathan, Sivakamasundari V, Hongbin Cao, Yuyan Jiang, Junfei Zhao, Deana R. Colburg, Patrick T. Neuhoefer, Yu Xu, Eyiwunmi E. Laseinde, Steven Artandi, Quynh-Thu Le. Terthighcells: key players in salivary gland homeostasis and regeneration after radiation therapy in adult mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5442.

Salivary gland homeostasis and regeneration after radiotherapy depend significantly on progenitor cells. However, the lineage of submandibular gland (SMG) progenitor cells remains less defined compared with other normal organs. Here, using a mouse strain expressing regulated CreERT2 recombinase from the endogenous Tert locus, we identify a distinct telomerase-expressing (TertHigh) cell population located in the ductal region of the adult SMG. These TertHighcells contribute to ductal cell generation during SMG homeostasis and to both ductal and acinar cell renewal 1 year after radiotherapy. TertHighcells maintain self-renewal capacity during in vitro culture, exhibit resistance to radiation damage, and demonstrate enhanced proliferative activity after radiation exposure. Similarly, primary human SMG cells with high Tert expression display enhanced cell survival after radiotherapy, and CRISPR-activated Tert in human SMG spheres increases proliferation after radiation. RNA sequencing reveals upregulation of “cell cycling” and “oxidative stress response” pathways in TertHighcells following radiation. Mechanistically, Tert appears to modulate cell survival through ROS levels in SMG spheres following radiation damage. Our findings highlight the significance of TertHighcells in salivary gland biology, providing insights into their response to radiotherapy and into their use as a potential target for enhancing salivary gland regeneration after radiotherapy.

Telomere length is an important biomarker of organismal aging and cellular replicative potential, but existing measurement methods are limited in resolution and accuracy. Here, we deploy digital telomere measurement by nanopore sequencing to understand how distributions of human telomere length change with age and disease. We measure telomere attrition and de novo elongation with unprecedented resolution in genetically defined populations of human cells, in blood cells from healthy donors and in blood cells from patients with genetic defects in telomere maintenance. We find that human aging is accompanied by a progressive loss of long telomeres and an accumulation of shorter telomeres. In patients with defects in telomere maintenance, the accumulation of short telomeres is more pronounced and correlates with phenotypic severity. We apply machine learning to train a binary classification model that distinguishes healthy individuals from those with telomere biology disorders. This sequencing and bioinformatic pipeline will advance our understanding of telomere maintenance mechanisms and the use of telomere length as a clinical biomarker of aging and disease.

Chimeric antigen receptor (CAR) T cell therapy has revolutionized oncology through engineered targeting of antigens on previously untreatable cancers. However, less than half of patients on CAR T cell therapy experience long-term disease control, with better outcomes observed in pediatric compared to adult populations. Senescent T cells have been shown to manifest defective killing abilities and the development of negative regulatory functions, with evidence suggesting that senescence may play a role in decreasing CAR T cell efficacy and persistence. Moreover, levels of telomerase have been shown to control the lifespan of human T cells, with increased levels delaying senescence. It has been shown that T cell exhaustion limits CAR T cell efficacy in the context of solid tumors, where CAR T cells have yet to demonstrate sustained responses. Using a model of CAR T cell exhaustion developed in our lab, and CAR T cells manufactured from younger and older donors, we have developed a comprehensive method to profile T cell functionality, phenotype, and proliferation. We are able to characterize features of senescence via cell surface markers, intracellular activity, telomere length, and telomerase activity as well as features of exhaustion. Using CRISPR/Cas9, we have inactivated TERT in primary human T cells and CAR T cells and interrogated the effects of hTERT knockout as well as of hTERT overexpression on key markers of T cell identity, exhaustion, senescence, and cytotoxic activity. Understanding telomerase in the context of CAR T cell therapy will provide mechanistic insights into senescence and exhaustion cellular programs and has the potential to inform increasingly effective CAR T cell cancer treatments. Citation Format: Tara Murty, Maria C. Ramello, Elena Sotillo, Lu Chen, Steven A. Artandi, Crystal L. Mackall. Exploring the role of telomerase in senescence and exhaustion in CAR T cell immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2815.

Tert-expressing cells have been identified within the acinar cell compartment of the pancreas in mice. Lineage tracing experiments indicate that these cells can maintain the exocrine compartment during homeostasis and repopulate the tissue during regeneration. Additional expression of mutant Kras in Tert-expressing acinar cells accelerates acinar clone formation and causes transdifferentiation to pre-invasive pancreatic intraepithelial neoplasms after injury. Here, using a genetic approach, we generated mice where tamoxifen injection leads to simultaneous deletion of TERT in Tert-expressing cells and activation of a reporter allele. We performed lineage tracing experiments to investigate the role of TERT in those cells. We studied the effect of acute deletion of TERT during homeostasis, regeneration and tumorigenesis. Acute deletion of TERT significantly inhibited the clone forming capability of Tert-expressing cells during homeostasis in pancreas after one year. To explore the requirement for TERT in an injury setting, we treated mice with cerulein to induce pancreatitis. Somatic inactivagion of TERT in Tert-expressing acinar stem cells impaired clone formation in mice treated with cerulein. To understand if loss of TERT affected transformation, we deleted TERT in acinar stem cells while specifically expressing activated Kras in these cells. Acute deletion of TERT in the presence of a mutant KrasG12D allele decreased the formation of metaplastic areas and PanIN lesions post-injury. These data suggest that a functional allele of Tert is necessary for the clone forming ability of Tert-expressing cells during homeostasis and regeneration as well as acinar to ductal metaplasia and PanIN formation in tumorigenesis. Citation Format: Patrick Neuhöfer, Stewart J. Kim, Gregory W. Charville, Steven E. Artandi. Somatic deletion of Tert inhibits clonal expansion of pancreatic acinar cell stem cells [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A076.

Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR/Cas9 and two adeno-associated viruses (AAV) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial basal stem cells (HBECs). The modified cells were enriched to obtain 60-80% tCD19 + UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

Promoting immune activation within the tumor microenvironment (TME) is a promising therapeutic strategy to reverse tumor immunosuppression and elicit anti-tumor immunity. To enable tumor-localized immunotherapy following intravenous administration, we chemically conjugated a polyspecific integrin-binding peptide (PIP) to an immunostimulant (Toll-like receptor 9 [TLR9] agonist: CpG) to generate a tumor-targeted immunomodulatory agent, referred to as PIP-CpG. We demonstrate that systemic delivery of PIP-CpG induces tumor regression and enhances therapeutic efficacy compared with untargeted CpG in aggressive murine breast and pancreatic cancer models. Furthermore, PIP-CpG transforms the immune-suppressive TME dominated by myeloid-derived suppressor cells into a lymphocyte-rich TME infiltrated with activated CD8(+)T cells, CD4(+)T cells, and B cells. Finally, we show that T cells are required for therapeutic efficacy and that PIP-CpG treatment generates tumor-specific CD8(+)T cells. These data demonstrate that conjugation to a synthetic tumor-targeted peptide can improve the efficacy of systemically administered immunostimulants and lead to durable anti-tumor immune responses.

Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.

Mutations in many genes that control the expression, the function, or the stability of telomerase cause telomere biology disorders (TBDs), such as dyskeratosis congenita, pulmonary fibrosis, and aplastic anemia. Mutations in a subset of the genes associated with TBDs cause reductions of the telomerase RNA moiety hTR, thus limiting telomerase activity. We have recently found that loss of the trimethylguanosine synthase TGS1 increases both hTR abundance and telomerase activity and leads to telomere elongation. Here, we show that treatment with the S-adenosylmethionine analog sinefungin inhibits TGS1 activity, increases the hTR levels, and promotes telomere lengthening in different cell types. Our results hold promise for restoring telomere length in stem and progenitor cells from TBD patients with reduced hTR levels.

Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5'-monomethylguanosine cap of small nuclear RNAs (snRNAs) to a trimethylguanosine cap. Here, we show that loss of TGS1 in Caenorhabditis elegans, Drosophila melanogaster and Danio rerio results in neurological phenotypes similar to those caused by survival motor neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3' tails that are often uridylated. snRNAs with defective 3' terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.

ABSTRACT Mouse lemurs are the smallest, fastest reproducing, and among the most abundant primates, and an emerging model organism for primate biology, behavior, health and conservation. Although much has been learned about their physiology and their Madagascar ecology and phylogeny, little is known about their cellular and molecular biology. Here we used droplet- and plate-based single cell RNA-sequencing to profile 226,000 cells from 27 mouse lemur organs and tissues opportunistically procured from four donors clinically and histologically characterized. Using computational cell clustering, integration, and expert cell annotation, we defined and biologically organized over 750 mouse lemur molecular cell types and their full gene expression profiles. These include cognates of most classical human cell types, including stem and progenitor cells, and the developmental programs for spermatogenesis, hematopoiesis, and other adult tissues. We also described dozens of previously unidentified or sparsely characterized cell types and subtypes. We globally compared cell type expression profiles to define the molecular relationships of cell types across the body, and explored primate cell and gene expression evolution by comparing mouse lemur cell transcriptomes to those of human, mouse, and macaque. This revealed cell type specific patterns of primate specialization, as well as many cell types and genes for which lemur provides a better human model than mouse. The atlas provides a cellular and molecular foundation for studying this primate model organism, and establishes a general approach for other emerging model organisms.

Telomerase is intimately associated with stem cells and upregulated in cancer, where it serves essential roles through its catalytic action in elongating telomeres, nucleoprotein caps that protect chromosome ends1. Overexpression of the telomerase reverse transcriptase (TERT) enhances cell proliferation through telomere-independent means, yet definitive evidence for such a direct role in stem cell function has yet to be revealed through loss-of-function studies. Here, we show that conditional deletion of TERT in spermatogonial stem cells (SSCs) markedly impairs competitive clone formation. Using lineage-tracing from the Tert locus, we find that TERT-expressing SSCs yield long-lived clones, but that selective TERT-inactivation in SSCs causes accelerated stem cell differentiation thereby disrupting clone formation. This requirement for TERT in clone formation is bypassed by expression of a catalytically inactive TERT transgene and occurs independently of the canonical telomerase complex. TERT inactivation induces a genome-wide reduction in open chromatin evident in purified SSCs, but not in committed progenitor cells. Loss of TERT causes reduced activity of the MYC oncogene and transgenic expression of MYC in TERT-deleted SSCs efficiently rescues clone formation. These data reveal a required catalytic activity-independent role for TERT in preventing stem cell differentiation, forge a genetic link between TERT and MYC and suggest new means by which TERT may promote tumorigenesis.

Background Tumor-localized delivery of Toll-like receptor (TLR) agonists is a promising strategy to promote immune activation within the tumor microenvironment (TME) to overcome tumor immunosuppression and induce anti-tumor immune responses. To enable localization to multiple tumor sites following systemic administration, we developed a fully-synthetic tumor-targeting TLR9 agonist and demonstrate its potential to transform the tumor immune microenvironment for effective tumor regression in mice. Methods An engineered synthetic peptide (PIP) that binds to multiple integrin receptors overexpressed in many solid tumors was chemically conjugated to a synthetic CpG oligonucleotide (TLR9 agonist), thereby generating a tumor-targeting immune-stimulant referred to as PIP-CpG. To facilitate clinical translation, PIP-CpG is cross-reactive between mouse, non-human primate, and human. Therapeutic studies were conducted in immune-competent mice bearing breast or pancreatic tumors to evaluate the efficacy of intravenously (IV)-injected PIP-CpG compared to IV-injected unmodified CpG or vehicle (PBS). We then performed mechanistic studies to evaluate the immune response elicited by PIP-CpG therapy. Results Intravenous dosing of PIP-CpG led to tumor regression and prolonged survival, and in some cases cures, relative to vehicle or unmodified CpG therapy in both murine breast (4T1) and pancreatic cancer (KPC-G2) models. This tumor regression was dependent on T cells as T cell depletion resulted in loss of therapeutic response. To study the effect of systemic therapy on the cellular landscape in the TME, we analyzed 4T1 breast tumors by flow cytometry. We found that vehicle and CpG IV-dosed mice had immunosuppressive TMEs comprised mostly of myeloid-derived suppressor cells (MDSCs; 43–68% of live cells) with minimal infiltrating T cells and B cells (5–16% of live cells). In contrast, the TME of PIP-CpG treated mice was transformed into a lymphocyte-rich "hot" tumor phenotype with massive infiltration by T cells and B cells (92–95% of live cells) and plummeting levels of MDSCs (down to ~1%). In addition, tumor-specific effector CD8+ T cells were generated in response to PIP-CpG therapy, but not CpG dosed IV, indicating that PIP-CpG therapy transforms the TME and elicits a T cell-mediated tumor-specific immune response. Furthermore, PIP-CpG was effective for treating MMTV-PyMT transgenic mice, which spontaneously develop multiple breast tumors. Murine toxicity studies indicated that effects of PIP-CpG were similar to CpG dosed IV or intratumorally, which have been well-tolerated in human clinical trials. Conclusions Tumor-directed systemic delivery of a TLR9 agonist transforms the TME via activated B and T cells and is promising for further development in patients with solid tumors. Ethics Approval All mouse experiments were performed in accordance with protocols approved by the Stanford Administrative Panel on Laboratory Animal Care.

Dyskeratosis congenita is a disease of impaired tissue maintenance downstream of telomere dysfunction. Characteristically, patients present with the clinical triad of nail dystrophy, oral leukoplakia, and skin pigmentation defects, but the disease involves degenerative changes in multiple organs. Mutations in telomere-binding proteins such as TINF2 (TRF1-interacting nuclear factor 2) or in telomerase, the enzyme that counteracts age related telomere shortening, are causative in dyskeratosis congenita. We present a patient who presented with severe hypoxemia at age 13. The patient had a history of myelodysplastic syndrome treated with bone marrow transplant at the age of 5. At age 18 she was hospitalized for an acute pneumonia progressing to respiratory failure, developed renal failure and ultimately, she and her family opted to withdraw support as she was not a candidate for a lung transplant. Sequencing of the patient's TINF2 locus revealed a heterozygous mutation (c.844C > T, Arg282Cys) which has previously been reported in a subset of dyskeratosis congenita patients. Tissue sections from multiple organs showed degenerative changes including disorganized bone remodeling, diffuse alveolar damage and small vessel proliferation in the lung, and hyperkeratosis with hyperpigmentation of the skin. Autopsy samples revealed a bimodal distribution of telomere length, with telomeres from donor hematopoietic tissues being an age-appropriate length and those from patient tissues showing pathogenic shortening, with the shortest telomeres in lung, liver, and kidney. We report for the first time a survey of degenerative changes and telomere lengths in multiple organs in a patient with dyskeratosis congenita.

The majority of the mammalian genome is transcribed into non-coding RNAs, many of which co-evolve with RNA-binding proteins (RBPs) to function as biochemically defined and tractable ribonucleoproteins (RNPs). Here, we applied ic SHAPE-a robust and versatile RNA structural probing pipeline-to endogenous RNPs purified from nuclei, providing base-resolution structural rationale for RNP activity and subcellular localization. Combining with genetic and biochemical reconstitutions, structural and functional alternations can be directly attributed to a given RBP without ambiguity. For complete details on the use and execution of this protocol, please refer to Chen et al. (2018).

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths worldwide1. Studies in human tissues and in mouse models have suggested that for many cancers, stem cells sustain early mutations driving tumour development2,3. For the pancreas, however, mechanisms underlying cellular renewal and initiation of PDAC remain unresolved. Here, using lineage tracing from the endogenous telomerase reverse transcriptase (Tert) locus, we identify a rare TERT-positive subpopulation of pancreatic acinar cells dispersed throughout the exocrine compartment. During homeostasis, these TERThigh acinar cells renew the pancreas by forming expanding clones of acinar cells, whereas randomly marked acinar cells do not form these clones. Specific expression of mutant Kras in TERThigh acinar cells accelerates acinar clone formation and causes transdifferentiation to ductal pre-invasive pancreatic intraepithelial neoplasms by upregulating Ras-MAPK signalling and activating the downstream kinase ERK (phospho-ERK). In resected human pancreatic neoplasms, we find that foci of phospho-ERK-positive acinar cells are common and frequently contain activating KRAS mutations, suggesting that these acinar regions represent an early cancer precursor lesion. These data support a model in which rare TERThigh acinar cells may sustain KRAS mutations, driving acinar cell expansion and creating a field of aberrant cells initiating pancreatic tumorigenesis.