Spatial Architecture of Myeloid and T Cells Orchestrates Immune Evasion and Clinical Outcome in Lung Cancer.

Understanding the role of the tumour microenvironment (TME) in lung cancer is critical to improving patient outcome. We identified four histology-independent archetype TMEs in treatment-naive early-stage lung cancer using imaging mass cytometry in the TRACERx study (n=81 patients/198 samples/2.3million cells). In immune-hot adenocarcinomas, spatial niches of T cells and macrophages increased with clonal neoantigen burden, whereas such an increase was observed for niches of plasma and B cells in immune-excluded squamous cell carcinomas (LUSC). Immune-low TMEs were associated with fibroblast barriers to immune infiltration. The fourth archetype, characterised by sparse lymphocytes and high tumour-associated neutrophil (TAN) infiltration, had tumour cells spatially separated from vasculature and exhibited low spatial intratumour heterogeneity. TAN-High LUSC had frequent PIK3CA mutations. TAN-High tumours harboured recently expanded and metastasis-seeding subclones and had a shorter disease-free survival independent of stage. These findings delineate genomic, immune and physical barriers to immune surveillance and implicate neutrophil-rich TMEs in metastasis.

Vitamin D regulates microbiome-dependent cancer immunity.

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis, which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.

Vitamin B5 supports MYC oncogenic metabolism and tumor progression in breast cancer.

Tumors are intrinsically heterogeneous and it is well established that this directs their evolution, hinders their classification and frustrates therapy1-3. Consequently, spatially resolved omics-level analyses are gaining traction4-9. Despite considerable therapeutic interest, tumor metabolism has been lagging behind this development and there is a paucity of data regarding its spatial organization. To address this shortcoming, we set out to study the local metabolic effects of the oncogene c-MYC, a pleiotropic transcription factor that accumulates with tumor progression and influences metabolism10,11. Through correlative mass spectrometry imaging, we show that pantothenic acid (vitamin B5) associates with MYC-high areas within both human and murine mammary tumors, where its conversion to coenzyme A fuels Krebs cycle activity. Mechanistically, we show that this is accomplished by MYC-mediated upregulation of its multivitamin transporter SLC5A6. Notably, we show that SLC5A6 over-expression alone can induce increased cell growth and a shift toward biosynthesis, whereas conversely, dietary restriction of pantothenic acid leads to a reversal of many MYC-mediated metabolic changes and results in hampered tumor growth. Our work thus establishes the availability of vitamins and cofactors as a potential bottleneck in tumor progression, which can be exploited therapeutically. Overall, we show that a spatial understanding of local metabolism facilitates the identification of clinically relevant, tractable metabolic targets.

WORKPLACE CLIMATE FOR SEXUAL AND GENDER MINORITIES IN ATHLETIC TRAINING.

CONTEXT: Sexual and gender minorities (SGM) are individuals with sexual orientations, gender identities and/or expressions that differ from cultural norms. SGMs often face workplace discrimination and report decreased physical and emotional well-being from discrimination. OBJECTIVE: To explore the workplace climate of SGM athletic trainers (AT). DESIGN: Sequential mixed-methods study. SETTING: Web-based survey and interviews. PATIENTS OR OTHER PARTICIPANTS: Criterion sampling of SGM ATs (117 survey participants and 12 interview participants). DATA COLLECTION AND ANALYSIS: We modified the LGBTQ Inclusion Assessment and the Organizational Self-Assessment for the survey and developed a semi-structured interview script (scale-level content validity index=0.94). We used means, standard deviations, frequencies and the consensual qualitative research tradition to characterize participant responses. Trustworthiness was established through reflexivity (researchers checking bias throughout the research process), member-checking, multi-analyst triangulation, internal and external auditing. RESULTS: Participants indicated their workplace was inclusive (24, 20.5%), somewhat inclusive (29, 24.8%), not inclusive (14, 12.0%), or did not indicate at all (50, 42.7%). Respondents most often indicated they were unsure of the stage of change their organizations and organizational units were in addressing LGBTQPIA+ issues in the workplace as well as specific actions taken for inclusion. Two domains emerged from the interview data: safety and inclusion. The safety domain represents aspects of the workplace climate that make the participants feel safe including organizational initiatives (12/12), patient-centered policies (7/12), local and federal regulations (7/12), and signaling (12/12). The inclusion domain represents how the participants felt a sense of belonging to the organization through their own experience (12/12), through the experiences of their patients (9/12), and through an infrastructure designed for inclusion (12/12). Participants expressed both the affirmative and the negative feelings of safety and inclusion throughout their responses. CONCLUSIONS: Organizations must take both structural and cultural actions to address the issues of exclusion and lack of safety.

The evolution of non-small cell lung cancer metastases in TRACERx.

Metastatic disease is responsible for the majority of cancer-related deaths1. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse.

Sports Medicine Physicians Comfort and Competence in Caring for Transgender and Gender Nonconforming Patients and Athletes.

OBJECTIVE: The purpose of this study was to explore primary care sports medicine physicians' comfort, competence, education, and scope of training in caring for transgender and gender nonconforming (TGNC) patients/athletes. DESIGN: Mixed-methods, cross-sectional survey. SETTING: Online. PATIENTS OR PARTICIPANTS: In total, 4300 e-mails were successfully sent with 252 eligible responses received from the American Medical Society for Sports Medicine members. INDEPENDENT VARIABLES: Previous relationships with TGNC persons; previous relationships with TGNC patients/athletes; frequency of care for TGNC patients/athletes. MAIN OUTCOME MEASURES: The participants completed a 38-item tool used to assess perceived comfort and competence treating TGNC patients/athletes. Physicians defined "transgender" and described their thoughts on unfair competitive advantage of transgender athletes. RESULTS: Most participants had worked with a TGNC patient (70.2%, n = 177), but far fewer worked with a TGNC athlete (n = 26.6%, n = 67). Among the participants who provided a definition of transgender (n = 183), only 28.4% (n = 52) of participants were able to correctly define the term, whereas most were able to partially (57.9%, n = 106) characterize the term. The most common mechanisms identified for learning about TGNC patients were reading peer-reviewed journal articles (44.8%, n = 113) and CME (41.3%, n = 104). Those with previous TGNC friend/family, patient, and athlete relationships had a significantly different level of comfort and competence treating TGNC patients/athletes. CONCLUSIONS: Previous care relationships with TGNC strongly influences comfort and perceived competence of primary care sports medicine physicians. Training, from unbiased peer-reviewed sources of data, is critical to improve care for TGNC patients/athletes.

GREM1 is required to maintain cellular heterogeneity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) shows pronounced epithelial and mesenchymal cancer cell populations1-4. Cellular heterogeneity in PDAC is an important feature in disease subtype specification3-5, but how distinct PDAC subpopulations interact, and the molecular mechanisms that underlie PDAC cell fate decisions, are incompletely understood. Here we identify the BMP inhibitor GREM16,7 as a key regulator of cellular heterogeneity in pancreatic cancer in human and mouse. Grem1 inactivation in established PDAC in mice resulted in a direct conversion of epithelial into mesenchymal PDAC cells within days, suggesting that persistent GREM1 activity is required to maintain the epithelial PDAC subpopulations. By contrast, Grem1 overexpression caused an almost complete 'epithelialization' of highly mesenchymal PDAC, indicating that high GREM1 activity is sufficient to revert the mesenchymal fate of PDAC cells. Mechanistically, Grem1 was highly expressed in mesenchymal PDAC cells and inhibited the expression of the epithelial-mesenchymal transition transcription factors Snai1 (also known as Snail) and Snai2 (also known as Slug) in the epithelial cell compartment, therefore restricting epithelial-mesenchymal plasticity. Thus, constant suppression of BMP activity is essential to maintain epithelial PDAC cells, indicating that the maintenance of the cellular heterogeneity of pancreatic cancer requires continuous paracrine signalling elicited by a single soluble factor.

USP25 promotes pathological HIF-1-driven metabolic reprogramming and is a potential therapeutic target in pancreatic cancer.

Deubiquitylating enzymes (DUBs) play an essential role in targeted protein degradation and represent an emerging therapeutic paradigm in cancer. However, their therapeutic potential in pancreatic ductal adenocarcinoma (PDAC) has not been explored. Here, we develop a DUB discovery pipeline, combining activity-based proteomics with a loss-of-function genetic screen in patient-derived PDAC organoids and murine genetic models. This approach identifies USP25 as a master regulator of PDAC growth and maintenance. Genetic and pharmacological USP25 inhibition results in potent growth impairment in PDAC organoids, while normal pancreatic organoids are insensitive, and causes dramatic regression of patient-derived xenografts. Mechanistically, USP25 deubiquitinates and stabilizes the HIF-1α transcription factor. PDAC is characterized by a severely hypoxic microenvironment, and USP25 depletion abrogates HIF-1α transcriptional activity and impairs glycolysis, inducing PDAC cell death in the tumor hypoxic core. Thus, the USP25/HIF-1α axis is an essential mechanism of metabolic reprogramming and survival in PDAC, which can be therapeutically exploited.

Substance Use Disorder Treatment, Perceived Need for Treatment, and Barriers to Treatment Among Parenting Women With Substance Use Disorder in US Rural Counties.

OBJECTIVE: Higher rates of substance use in rural counties compared to urban counties have been well documented. Low perceived need for treatment among those with substance use disorder (SUD) has also been documented in the literature. However, not much is known about SUD treatment among parenting women in rural counties and the impact of perceived need for treatment in seeking care. Little research has also examined barriers to SUD treatment among parenting women in rural communities. METHODS: Using a large nationally representative dataset, the study utilizes multivariable logistic regression models to estimate the differences in utilizing SUD treatment among parenting women with SUD in rural and urban counties in the United States. Role of perceived need for SUD treatment and barriers related to finance, access, and stigma are also examined. RESULTS: Parenting women in rural counties with SUD who perceive a need for treatment have more than 90% lower odds of receiving treatment compared to those in urban counties. In addition, parenting women with SUD in rural counties have more than 50% higher odds of identifying access-related issues such as lack of openings in programs, unavailability of treatment facilities, and lack of transportation as barriers to care compared to parenting women in urban counties. CONCLUSION: Diagnosis of SUD among parenting women is steadily increasing in rural communities. While many resources in combatting maternal SUD are being utilized, policy and programmatic responses tailored for mothers with SUD in rural communities might help increase utilization of treatment and reduce barriers to treatment.

USP28 deletion and small-molecule inhibition destabilizes c-MYC and elicits regression of squamous cell lung carcinoma.

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient's 5-year survival rate is less than 5%. The ubiquitin-specific protease 28 (USP28) has been implicated in tumourigenesis through its stabilization of the oncoproteins c-MYC, c-JUN, and Δp63. Here, we show that genetic inactivation of Usp28-induced regression of established murine LSCC lung tumours. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-MYC, c-JUN, and Δp63 proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumours and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.

Functional antibody and T-cell immunity following SARS-CoV-2 infection, including by variants of concern, in patients with cancer: the CAPTURE study.

Patients with cancer have higher COVID-19 morbidity and mortality. Here we present the prospective CAPTURE study (NCT03226886) integrating longitudinal immune profiling with clinical annotation. Of 357 patients with cancer, 118 were SARS-CoV-2-positive, 94 were symptomatic and 2 patients died of COVID-19. In this cohort, 83% patients had S1-reactive antibodies, 82% had neutralizing antibodies against WT, whereas neutralizing antibody titers (NAbT) against the Alpha, Beta, and Delta variants were substantially reduced. Whereas S1-reactive antibody levels decreased in 13% of patients, NAbT remained stable up to 329 days. Patients also had detectable SARS-CoV-2-specific T cells and CD4+ responses correlating with S1-reactive antibody levels, although patients with hematological malignancies had impaired immune responses that were disease and treatment-specific, but presented compensatory cellular responses, further supported by clinical. Overall, these findings advance the understanding of the nature and duration of immune response to SARS-CoV-2 in patients with cancer.

JunD, not c-Jun, is the AP-1 transcription factor required for Ras-induced lung cancer.

The AP-1 transcription factor c-Jun is required for Ras-driven tumorigenesis in many tissues and is considered as a classical proto-oncogene. To determine the requirement for c-Jun in a mouse model of K-RasG12D-induced lung adenocarcinoma, we inducibly deleted c-Jun in the adult lung. Surprisingly, we found that inactivation of c-Jun, or mutation of its JNK phosphorylation sites, actually increased lung tumor burden. Mechanistically, we found that protein levels of the Jun family member JunD were increased in the absence of c-Jun. In c-Jun-deficient cells, JunD phosphorylation was increased, and expression of a dominant-active JNKK2-JNK1 transgene further increased lung tumor formation. Strikingly, deletion of JunD completely abolished Ras-driven lung tumorigenesis. This work identifies JunD, not c-Jun, as the crucial substrate of JNK signaling and oncogene required for Ras-induced lung cancer.

Immunogenomics of Colorectal Cancer Response to Checkpoint Blockade: Analysis of the KEYNOTE 177 Trial and Validation Cohorts.

BACKGROUND & AIMS: Colorectal cancer (CRC) shows variable response to immune checkpoint blockade, which can only partially be explained by high tumor mutational burden (TMB). We conducted an integrated study of the cancer tissue and associated tumor microenvironment (TME) from patients treated with pembrolizumab (KEYNOTE 177 clinical trial) or nivolumab to dissect the cellular and molecular determinants of response to anti- programmed cell death 1 (PD1) immunotherapy. METHODS: We selected multiple regions per tumor showing variable T-cell infiltration for a total of 738 regions from 29 patients, divided into discovery and validation cohorts. We performed multiregional whole-exome and RNA sequencing of the tumor cells and integrated these with T-cell receptor sequencing, high-dimensional imaging mass cytometry, detection of programmed death-ligand 1 (PDL1) interaction in situ, multiplexed immunofluorescence, and computational spatial analysis of the TME. RESULTS: In hypermutated CRCs, response to anti-PD1 immunotherapy was not associated with TMB but with high clonality of immunogenic mutations, clonally expanded T cells, low activation of Wnt signaling, deregulation of the interferon gamma pathway, and active immune escape mechanisms. Responsive hypermutated CRCs were also rich in cytotoxic and proliferating PD1+CD8 T cells interacting with PDL1+ antigen-presenting macrophages. CONCLUSIONS: Our study clarified the limits of TMB as a predictor of response of CRC to anti-PD1 immunotherapy. It identified a population of antigen-presenting macrophages interacting with CD8 T cells that consistently segregate with response. We therefore concluded that anti-PD1 agents release the PD1-PDL1 interaction between CD8 T cells and macrophages to promote cytotoxic antitumor activity.

Functional antibody and T cell immunity following SARS-CoV-2 infection, including by variants of concern, in patients with cancer: the CAPTURE study.

Patients with cancer have higher COVID-19 morbidity and mortality. Here we present the prospective CAPTURE study, integrating longitudinal immune profiling with clinical annotation. Of 357 patients with cancer, 118 were SARS-CoV-2 positive, 94 were symptomatic and 2 died of COVID-19. In this cohort, 83% patients had S1-reactive antibodies and 82% had neutralizing antibodies against wild type SARS-CoV-2, whereas neutralizing antibody titers against the Alpha, Beta and Delta variants were substantially reduced. S1-reactive antibody levels decreased in 13% of patients, whereas neutralizing antibody titers remained stable for up to 329 days. Patients also had detectable SARS-CoV-2-specific T cells and CD4+ responses correlating with S1-reactive antibody levels, although patients with hematological malignancies had impaired immune responses that were disease and treatment specific, but presented compensatory cellular responses, further supported by clinical recovery in all but one patient. Overall, these findings advance the understanding of the nature and duration of the immune response to SARS-CoV-2 in patients with cancer.

Providing Transgender Patient Care: Athletic Trainers' Compassion and Lack of Preparedness.

CONTEXT: Previous researchers have indicated that athletic trainers (ATs) had a favorable view of treating transgender patients, yet the ATs did not perceive themselves as competent in their patient care knowledge or abilities. OBJECTIVE: To gain more in-depth information about ATs' knowledge and experiences regarding the health care needs of transgender student-athletes. DESIGN: Mixed-methods study. SETTING: Individual, semistructured follow-up interviews. PATIENTS OR OTHER PARTICIPANTS: Fifteen ATs (4 men, 10 women, 1 transgender female; age = 34 ± 9 years, experience = 11 ± 8 years) who took part in a cross-sectional survey in April 2018. MAIN OUTCOME MEASURE(S): The interviews were audio recorded and transcribed verbatim. Member checking was completed to ensure trustworthiness of the data. Next, the data were analyzed via a multiphase process and 3-member coding team who followed the consensual qualitative research tradition. The coding team analyzed the transcripts for domains and categories. The final consensus codebook and coded transcripts were audited by a member of the research team for credibility. RESULTS: Four main domains were identified: (1) perceived deficiencies, (2) misconceptions, (3) concerns, and (4) creating safety. Participants described knowledge deficiencies in themselves, health care providers within their units, and providers able to provide safe transition care. The ATs demonstrated misconceptions when defining transgender and transitioning and when describing how the body responds to hormone replacement therapy. They expressed concern for the mental health and wellness, self-image, and potential cost of transgender health care for transgender student-athletes. However, participants also described efforts to create safety within their units by validating transgender patients, instilling trust, adjusting the physical environment, and engaging in professional development to improve their knowledge. CONCLUSIONS: Athletic trainers wanted to create a safe space for transgender student-athletes but lacked the necessary knowledge to treat transgender patients. Professional resources to improve their knowledge, skills, and abilities in caring for transgender patients are a continuing need.

Athletic Trainers' Competence, Education, and Perceptions Regarding Transgender Student-Athlete Patient Care.

CONTEXT: Transgender student-athletes are increasingly participating in sport, requiring athletic trainer (AT) preparedness to care for their needs. OBJECTIVE: To measure ATs' (1) perceived definition of transgender, (2) comfort and competence working with transgender student-athletes, (3) sources of education, (4) perceived legal concerns, and (5) perception of competitive advantage. DESIGN: Cross-sectional study. SETTING: Mixed-methods survey. PATIENTS OR OTHER PARTICIPANTS: Collegiate or university ATs (n = 5537) received an email invitation to participate; the data of 667 ATs were included in the analysis. MAIN OUTCOME MEASURE(S): A multipart 43-item questionnaire addressing the primary objectives of the study, with other factors that were explored in relation to these objectives to uncover potential influences on their responses. We calculated descriptive statistics, and for open-ended responses, we used the consensual qualitative research tradition. RESULTS: About half (48.1%, n = 321) of the participants agreed they were competent in treating transgender patients, but only 36.0% (n = 240) believed they were competent in practicing collaboratively with an endocrinologist in the drug-screening processes. Fewer than half (45.6%, n = 304) of participants felt they were competent in using appropriate terminology relating to transgender patients. The ATs disagreed when asked if they were competent regarding counseling transgender patients about the effects of hormone replacement therapy on sport participation (48.1%, n = 321) or on mental health concerns (40.3%, n = 269). Participants learned most frequently from media outlets (35.2%, n = 235) or personal experiences with family, friends, or themselves (33.7%, n = 225), yet 35.1% (n = 243) received no education in caring for transgender patients. Many ATs (41.2%, n = 278) believed that transgender female student-athletes had a competitive advantage. In contrast, 6.6% (n = 44) of participants indicated that transgender male student-athletes had a competitive advantage. CONCLUSIONS: Although collegiate ATs generally felt competent in treating transgender patients, they did not feel capable of addressing specific aspects of transgender patients' health care needs. Regardless of the resulting perceived unfair advantage, ATs must be aware of the regulations and therapeutic effects associated with hormone-related therapy for transgender student-athletes.

The Transcription Co-Repressors MTG8 and MTG16 Regulate Exit of Intestinal Stem Cells From Their Niche and Differentiation Into Enterocyte vs Secretory Lineages.

BACKGROUND & AIMS: Notch signaling maintains intestinal stem cells (ISCs). When ISCs exit the niche, Notch signaling among early progenitor cells at position +4/5 regulates their specification toward secretory vs enterocyte lineages (binary fate). The transcription factor ATOH1 is repressed by Notch in ISCs; its de-repression, when Notch is inactivated, drives progenitor cells to differentiate along the secretory lineage. However, it is not clear what promotes transition of ISCs to progenitors and how this fate decision is established. METHODS: We sorted cells from Lgr5-GFP knockin intestines from mice and characterized gene expression patterns. We analyzed Notch regulation by examining expression profiles (by quantitative reverse transcription polymerase chain reaction and RNAscope) of small intestinal organoids incubated with the Notch inhibitor DAPT, intestine tissues from mice given injections of the γ-secretase inhibitor dibenzazepine, and mice with intestine-specific disruption of Rbpj. We analyzed intestine tissues from mice with disruption of the RUNX1 translocation partner 1 gene (Runx1t1, also called Mtg8) or CBFA2/RUNX1 partner transcriptional co-repressor 3 (Cbfa2t3, also called Mtg16), and derived their organoids, by histology, immunohistochemistry, and RNA sequencing (RNA-seq). We performed chromatin immunoprecipitation and sequencing analyses of intestinal crypts to identify genes regulated by MTG16. RESULTS: The transcription co-repressors MTG8 and MTG16 were highly expressed by +4/5 early progenitors, compared with other cells along crypt-villus axis. Expression of MTG8 and MTG16 were repressed by Notch signaling via ATOH1 in organoids and intestine tissues from mice. MTG8- and MTG16-knockout intestines had increased crypt hyperproliferation and expansion of ISCs, but enterocyte differentiation was impaired, based on loss of enterocyte markers and functions. Chromatin immunoprecipitation and sequencing analyses showed that MTG16 bound to promoters of genes that are specifically expressed by stem cells (such as Lgr5 and Ascl2) and repressed their transcription. MTG16 also bound to previously reported enhancer regions of genes regulated by ATOH1, including genes that encode Delta-like canonical Notch ligand and other secretory-specific transcription factors. CONCLUSIONS: In intestine tissues of mice and human intestinal organoids, MTG8 and MTG16 repress transcription in the earliest progenitor cells to promote exit of ISCs from their niche (niche exit) and control the binary fate decision (secretory vs enterocyte lineage) by repressing genes regulated by ATOH1.

CD9 identifies pancreatic cancer stem cells and modulates glutamine metabolism to fuel tumour growth.

Pancreatic ductal adenocarcinoma (PDAC) shows great cellular heterogeneity, with pronounced epithelial and mesenchymal cancer cell populations. However, the cellular hierarchy underlying PDAC cell diversity is unknown. Here we identify the tetraspanin CD9 as a marker of PDAC tumour-initiating cells. CD9high cells had increased organoid formation capability, and generated tumour grafts in vivo at limiting dilutions. Tumours initiated from CD9high cells recapitulated the cellular heterogeneity of primary PDAC, whereas CD9low cells produced only duct-like epithelial progeny. CD9 knockdown decreased the growth of PDAC organoids, and heterozygous CD9 deletion in Pdx1-Cre; LSL-KRasG12D; p53F/F mice prolonged overall survival. Mechanistically, CD9 promoted the plasma membrane localization of the glutamine transporter ASCT2, enhancing glutamine uptake in PDAC cells. Thus, our study identifies a PDAC subpopulation capable of initiating PDAC and giving rise to PDAC heterogeneity, suggesting that the cellular diversity of PDAC is generated by PDAC stem cell differentiation.

ATMIN Is a Tumor Suppressor Gene in Lung Adenocarcinoma.

Tumor cells proliferate rapidly and thus are frequently subjected to replication stress and the risk of incomplete duplication of the genome. Fragile sites are replicated late, making them more vulnerable to damage when DNA replication fails to complete. Therefore, genomic alterations at fragile sites are commonly observed in tumors. FRA16D is one of the most common fragile sites in lung cancer, however, the nature of the tumor suppressor genes affected by FRA16D alterations has been controversial. Here, we show that the ATMIN gene, which encodes a cofactor required for activation of ATM kinase by replication stress, is located close to FRA16D and is commonly lost in lung adenocarcinoma. Low ATMIN expression was frequently observed in human lung adenocarcinoma tumors and was associated with reduced patient survival, suggesting that ATMIN functions as a tumor suppressor in lung adenocarcinoma. Heterozygous Atmin deletion significantly increased tumor cell proliferation, tumor burden, and tumor grade in the LSL-KRasG12D; Trp53 F/F (KP) mouse model of lung adenocarcinoma, identifying ATMIN as a haploinsufficient tumor suppressor. ATMIN-deficient KP lung tumor cells showed increased survival in response to replication stress and consequently accumulated DNA damage. Thus, our data identify ATMIN as a key gene affected by genomic deletions at FRA16D in lung adenocarcinoma. SIGNIFICANCE: These findings identify ATMIN as a tumor suppressor in LUAD; fragility at chr16q23 correlates with loss of ATMIN in human LUAD and deletion of Atmin increases tumor burden in a LUAD mouse model.