ULK1 Knockout suppresses Pancreatic Cancer Progression by Inhibiting Autophagy and Enhancing Anti-tumor Immunity

ULK1 Knockout suppresses Pancreatic Cancer Progression by Inhibiting Autophagy and Enhancing Anti-tumor Immunity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article ULK1 Knockout suppresses Pancreatic Cancer Progression by Inhibiting Autophagy and Enhancing Anti-tumor Immunity Heesun Cheong, Hana Jeong, Jinju Lee, Jiyoon Son, Jinkyung Lee, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6438501/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Dec, 2025 Read the published version in Experimental & Molecular Medicine → Version 1 posted 9 You are reading this latest preprint version Abstract Autophagy plays a dual role in cancer, acting as a tumor suppressor and promoter depending on tumor stage and context. While core autophagy genes such as Atg5 and Atg7, the role of Unc-51-like kinase 1 (ULK1) —a key autophagy initiator-remains poorly understood in pancreatic ductal adenocarcinoma (PDAC). In this study, we investigated the role of ULK1 using tissue-specific deletion in GEM models. Although ULK1 mRNA levels remained unchanged between normal and tumor cells in The Cancer Genome Atlas (TCGA) dataset, multiplex immunohistochemistry revealed elevated ULK1 activity, marked by phosphorylated ATG14, in high-grade human PDAC tissues. Genetic deletion of Ulk1 impaired autophagy, reduced cell proliferation, colony formation, and invasiveness of pancreatic cancer cells. In vivo both syngeneic orthotopic and KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre) mouse model with tissue specific Ulk1 deletion exhibited significant delayed tumor progression, reduced tumor burden, and extended survival. Importantly, Ulk1 deficiency remodeled the tumor immune microenvironment by reducing tumor-promoting polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and neutrophils while enhancing recruitment of cytotoxic CD8+ T cells and MHC-II+ antigen-presenting cells. Chemokine and cytokine profiling revealed that downregulation of Cxcl2, Ccl2, and G-CSF, might acting for PMN-MDSCs and neutrophils recruitment and survival, with concurrent upregulation of GM-CSF for dendritic cell infiltration, thereby inducing antitumor immunity. These findings provide novel insights into the role of ULK1 in PDAC progression through tumor-intrinsic metabolic support by autophagy activation and immune modulation by tumor-derived cytokines. Targeting ULK1 may represent a promising therapeutic strategy by inhibiting autophagy and enhancing antitumor immune responses in pancreatic cancer. Biological sciences/Cancer/Cancer models Biological sciences/Cell biology/Autophagy/Macroautophagy Health sciences/Diseases/Cancer/Tumour immunology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Autophagy is a conserved intracellular degradation pathway that sustains cellular homeostasis by recycling macromolecules and clearing damaged organelles. Autophagy plays a crucial role in balancing normal cell functions and multiple pathophysiological conditions, including cancer. While it preserves genome stability in early cancer, it later supports tumor survival by maintaining organelles, enabling recycling, and fulfilling metabolic demands (1, 2). The tumor-promoting role of autophagy has been validated in genetically engineered mouse (GEM) models. Oncogenic mutations in HRas or KRas increase basal autophagy, which is necessary for tumor cell survival during tumorigenesis (3). Deletion of Atg5 or Atg7 in KRas G12D -driven spontaneous lung cancer models reduced tumor size compared with that in wild-type (WT) mice (4, 5). Melanomas with BRAF mutations (6) and pancreatic cancers harboring 95% KRas mutations also exhibit high dependency on autophagy genes such as Atg5 and Atg7 (7, 8). In addition, depleting Atg proteins like FIP200, Atg16, or Atg4 suppresses tumor growth in various cancers (9-11). Accordingly, autophagy inhibition has been considered a promising therapeutic strategy (7, 8). Unc-51-like kinase 1 (ULK1), a serine/threonine kinase, has gained attention as a druggable target, which plays a crucial role in the initiation step of autophagy (12). ULK1 forms a complex with proteins such as ATG13, ATG101, and FIP200/RB1CC1, facilitated by the phosphorylation of ATG13 (13-15), and subsequently phosphorylating the components of the Class III PI(3) Kinase/Vps34 complex, including ATG14 and Beclin1 to drive autophagosome formation (16-18). ULK1 activity is tightly controlled by upstream nutrient-signaling molecules such as mTORC1 and AMPK, which phosphorylate ULK1 at distinct residues to either inhibit or enhance its function (19). The ULK family includes ULK1, ULK2, ULK3, ULK4, and STK368, with ULK1 and ULK2 showing the highest similarity (20). However, ULK1 is the dominant initiator of autophagy, as seen in Ulk1 −/− Ulk2 −/− double-knockout (DKO) mice, which exhibit severe neonatal mortality and autophagy defects similar to other core Atg gene deletions (21-24). Although the cellular functions of ULK1 have been studied in several diseases, direct evidence of ULK1 role on tumor progression of PDAC, remains largely unexplored. Given the significant dependency of KRas-driven tumors including PDAC on autophagy (4, 7, 8, 25), pharmacological or genetic inhibition of KRas-downstream RAF, MEK, and ERK pathways has been shown to enhance autophagy, which supports the rationale for combining MAP kinase inhibitors with a derivative of chloroquine (CQ), a lysosomotropic agent (26-28). However, the therapeutic potential of CQ derivatives limited by the requirement for high inhibitory doses and poor selectivity, highlighting the need for more potent and selective autophagy modulators. Targeting ULK1, an autophagy-initiating kinase, has emerged as an alternative strategy for autophagy inhibition. A series of ULK1 inhibitors have demonstrated antitumor effects in vitro and in xenograft models (29, 30), and selective ULK1 inhibitors have shown synergistic tumor regression when combined with KRas or downstream effector inhibitors (31). However, despite the increasing interest in ULK1-targeted therapies, the exact role of ULK1 in cancer progression remains poorly understood. In this study, we investigate how ULK1 function influences PDAC progression by employing syngeneic orthotopic and spontaneous cancer GEM models. Our findings reveal that tumor-intrinsic ULK1 deletion suppresses PDAC progression by impairing autophagy-mediated tumor adaptation and by reprogramming the tumor microenvironment through altered infiltration of distinct immune cell subtypes, thereby providing tumor-suppressive immune states. These results provide novel insights into ULK1 as a promising therapeutic target in pancreatic cancer. Materials and Methods Cell lines MIA PaCa-2 and HEK293T cells were kindly provided by Drs. Yongdoo Choi and Jong Heon Kim (National Cancer Center Korea; NCC Korea), which are originally purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). KPC cell lines generous gift from Jong Heon Kim (NCC Korea), which are originally purchased from the Ximbio (London, UK), and were derived from PDAC tumors arising in KPC mice on a C57BL/6 background. All cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS; HyClone, Logan, UT), 100 U/mL penicillin, and 100 μg/mL of streptomycin (Gibco, Grand Island, NY), and maintained at 37 ℃ in a humidified incubator with 5 % CO2. For amino acid starvation media (-AA), Earle’s Balanced Salt Solution (EBSS, HyClone) or Hank’s balanced saline solution (HBSS) was supplemented with 10 % dialyzed FBS, glucose, vitamins, HEPES, and minerals at the same concentrations as in DMEM. Generation of stable cell lines CRISPR-Cas9-mediated knockout of Ulk1 was performed lentiCRISPR v2 vector (a gift from Feng Zhang ;Addgene plasmid # 52961; RRID: Addgene_52961) (32). sgRNA oligo targeting mouse U lk1 CRISPR-Cas9 guide RNA (U0448BI200-1) or non-target sequence (sgControl) were synthesized (Gene Script, Piscataway, NJ). The guide RNA sequence were selected using the CRISPICK online tool (Broad institute, https://portals.broadinstitute.org/gppx/crispick/public) and sgRNA sequences are listed in Supplementary Table S2. Viral transduction processes were followed by standard protocols detailed in supplementary materials and methods. Reagents Hoechst 33342 (H3570) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Rapamycin (R0395) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell proliferation and Viability assay Cell proliferation was monitored using the image-based cell proliferation analyzer IncuCyte TM (Essen Instruments, Ann Arbor, MI, USA). Cells were seeded in complete DMEM media and imaged throughout the indicated time period. IncuCyte TM automated cell proliferation detector was used to measure cell confluence over time. Cell viability was determined by Annexin V and PI staining following standard protocols at the indicated time periods (556547; BD Biosciences, San Jose, CA, USA), and analyzed using the FACS Verse analyzer (BD Biosciences). Viability was assessed based on double-negative populations, and dead cells were defined by Annexin V⁺ and/or PI⁺ staining. Colony formation and Spheroid cultures KPC cells (5 x 10 2 cells / well) were seeded in 12-well plates in complete media (10 % FBS in DMEM). After 7 days, cells were fixed for 10 mins in 10 % formalin (HT501128-4L; Sigma-Aldrich) and stained with 0.25 % crystal violet (C6158; Sigma-Aldrich). For 3D spheroids culture, KPC cells (1 x 10 3 cells /well) were seeded in 96-well round-bottom Ultra Low Attachment (ULA) plates (7007; Corning Inc., Corning, NY) mixed with 1 % Matrigel (354234; Corning Inc.) and complete media. Spheroid growth was monitored for 7 days and imaged using HCS system Operetta CLS (PerkinElmer, Waltham, MA). Quantification was performed by using Harmony software (PerkinElmer). Cell I nvasion assay Invasion assay was performed to ascertain cell invasion using 0.8 μm Transwell apparatus (Corning Inc.) with coated Matrigel (354234; Corning Inc.) on a 24-well plate. KPC cells (1 × 10 5 ) suspended in 100 μl serum-free medium were seeded into the upper chamber and complete DMEM was added to the lower chamber. After incubation at 37 °C for 24 h, invaded cells were stained using Diff-Quik reagents (Sysmex Corporation, Kobe, Japan) and counted under an inverted microscope at a 4 × and 20 × magnifications. LSL - Kras G12D/+ ; LSL - Trp53 R172H/+ ; Pdx1-Cre ; Ulk1 fl / fl Genetically Engineered Mouse Model Kras G12D/+ (B6.129S4- Kras tm4Tyj , strain #01XJ6) was received from the NCI mouse repository (NCI, Bethesda, MD, USA). Trp53 R172H/+ (129S- Trp53 tm2Tyj /J, strain #008652) and Pdx1 -Cre (B6.FVB-Tg ( Pdx1 -Cre)6Tuv/J, strain #014647) mouse were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). These mice were backcrossed more than six times with C57BL/6 and were subsequently used for generating KPC ( Kras G12D/+ ; Trp53 R172H/+ ; Pdx1 -Cre) mouse. The KPC model of PDAC was first described in 2005 and incorporates, through Cre-lox technology, the conditional activation of mutant endogenous alleles of the Kras and Trp53 gene (33). Ulk1 fl / fl (B6.129- Ulk1 tm1Thsn /J, stock #017976) mice were purchased from The Jackson Laboratory (Sacramento, CA), which were generated and donated by Craig B. Thompson (Memorial Sloan Kettering Cancer Center, NY, USA) (24). The Ulk1 fl/fl and KPC mice were crossed, and after several rounds of breeding, KPC ; Ulk1 fl/fl mice were successfully obtained. All animal procedures were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the NCC. NCC is an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International)-accredited facility that abides by the guidelines of the Institute of Laboratory Animal Resources (ILAR) Guide and Usage Committee. The methods applied in this study were performed in accordance with the approved guidelines. Orthotopic P ancreatic C ancer mouse model Six-week-old female C57BL/6 mice (Orient Bio Inc., Seongnam, Korea) were orthotopically injected with 2 × 10⁵ KPC sgSC or KPC sg Ulk1 cells suspended in 1:1 Matrigel (50 µL) into the pancreatic parenchyma. After injection, the peritoneum and skin were closed with a 6-0 suture (639G; Ethicon, Raritan, NJ). At 3 weeks later of allograft, pancreas was isolated from the mice and used for indicated experiments. Histological and Immunohistochemical Analyses Pancreas tissues from indicated allografted mice or pancreatic tissues from KPC ; Ulk1 +/fl and KPC ; Ulk1 fl/fl GEM mice were isolated from 12 weeks to 17 weeks old mice, respectively. The tissues were fixed in 10% Neutral Buffered Formalin (BN019, Biosolution, Seoul, Korea) and paraffin-embedded. Section (4 μm thick) of mouse pancreas were deparaffinized, rehydrated and incubated in boiling CC1(pH 9) or CC2(pH 6) for antigen retrieval by Benchmark TX (950-123 or 124; Ventana Medical Systems, Oro Valley, AZ) or Discovery XT (Ventana Medical Systems). Immunohistochemical (IHC) staining was performed with the indicated primary antibodies (Supplementary Table S1) and then detected with a DAB detection kit (Ventana Medical Systems) according to the manufacturer’s instructions, followed by counterstaining with hematoxylin (Ventana Medical Systems). The stained images were acquired using the Vectra Polaris (Perkin Elmer/AKOYA Biosciences) and quantified using inForm ⓡ Tissue Analysis Software (AKOYA Biosciences, Marlborough, MA). H-Score = (1 X % of 1+ staining spot) + (2 X % of 2+ staining spot) + (3 X % of 3+ staining spot). Statistical analysis was calculated using Graphpad Prism 8.0.1. Multiplex I mmunohistochemistry (multi-IHC) assay TMA sections from PDAC patients (PA241e, TissueArray.Com LLC, Derwood, MD, USA) and the FFPE pancreas tissue from KPC Ulk1 +/fl and KPC Ulk1 fl/fl mice were subjected to multiplex immunohistochemistry (multi-IHC) with indicated antibodies (Supplementary Table S1) and automated staining Leica Bond RX (Leica Biosystems, Vista, CA) using Opal 7 kit (#NEL871001KT, #ARR1001KT; Akoya Biosciences). Tyramide signal amplification (TSA) fluorophores were used to combine with each antibody separately according to manufacturer’s instructions. Imaging and quantification were performed by Vectra Polaris Automated Quantitative Pathology Imaging System (Perkin Elmer Inc.) and inForm image analysis software (Akoya Biosciences). Data were compiled and analyzed after it merged and consolidated each case by R studio (version 2021.09.2.0). Flow C ytometry for Tumor Immunophenotyping For the immunophenotyping of tumor, tumor tissues were dissociated in a fully automated way by using the gentleMACS TM Octo Dissociator with Heaters (130-096-427; Miltenyi Biotec, Bergisch Gladbach, Germany) with collagenase P (C7657, Merk) and the Tumor Dissociation Kit (130-096-730; Miltenyi Biotec), which are optimized for epitope preservation. Digested tissues were filtered through a 70 μm nylon cell strainer (352350; BD Falcon, Franklin Lakes, NJ). Cells were blocked with anti-mouse CD16/CD32 antibody (Mouse BD Fc Block TM ) (BD553142, clone 2.4G2; BD Biosciences) and stained with indicated antibodies for 20 min at 4 °C. Intracellular staining was performed using Fixation/Permeabilization Kit (BD554714; BD Bioscience). Samples were analyzed on a LSRFortessa™ Cell Analyzer (BD Biosciences). Cytokine array Cytokine profiles were assessed using the Proteome Profiler Mouse Cytokine Array Kit (ARY006; R&D systems, Minneapolis, MN, USA) following the manufacturer’s instruction. Detailly, mouse pancreatic cancer tissues were homogenized by Tissue Lyser II (QIAGEN, Hilden, Germany) in PBS containing protease inhibitor cocktail (Roche, Basel, Switzerland). and then lysed with 1% Triton X-100. Cellular debris was removed by centrifugation at 10,000xg for 5 min at 4 ℃ and protein was quantified using BCA Protein Assay (23227; Thermo Fisher Scientific Inc.). Conditioned media were collected from cells cultured under identical conditions for 24 hours. Media were then normalized to the total cellular protein content, as determined from corresponding cell lysates. Membranes incubated with conditioned media were developed and visualized using the FUSION SOLO S imaging system (Vilber, Collégien, France) with exposure times ranging from 2 to 6 minutes. Immune C ell V iability analysis Pancreas, spleen and blood from the abdominal aorta were collected and dissociated as indicated above. PMN-MDSC/neutrophil (CD45 + CD11b + Ly6G + ), CD8 T cell (CD45 + CD3 + CD8 + ) and DCs (CD45 + CD11c + ) were sorted using Melody (BD Biosciences), and then cultured in the presence of 50% conditioned media from KPC sgSC or KPC_sg Ulk1 cells on a 384- or 96-well black well plate (6057308 and 6055302; PerkinElmer). After incubation with indicated time, live cell numbers were assessed using Calcein-AM (C1430; Invitrogen, Carlsbad, CA) staining. Imaging and quantification were performed using the Operetta CLS system and Harmony software (PerkinElmer). Bioinformatic Data processing The Cancer Genome Atlas (TCGA)-pancreatic adenocarcinoma (PAAD) patients were stratified with the autophagy score using the surv_cutpoint function from the survminer package (version 0.4.9). Kaplan-Meier overall survival plot and the p-value between patient groups was calculated using the Log-Rank test implemented in the survival package (version 3.5-7). Cox Hazard ratio and confidence interval was calculated using coxph function of the survival package. The autophagy score was calculated using a modified version of ssGSEA2.0, which is an adaptation of the GSEA algorithm for single-cell analysis (34, 35). The list of genes used for the autophagy score calculation can be found in the supplementary information. Default parameters for ssGSEA were used to calculate scores for TCGA-PAAD patients and for cells in single-cell data from pancreatic cancer patients (CRA001160) (35, 36). Data availability For gene set enrichment analysis, TCGA database were accessed in the Cancer Integrative Platform (cBioportal; http://www.cBioportal.mskcc.org/). For the survival analysis, the latest TCGA iteration of transcriptome and clinical data was downloaded directly using the application programming interface provided by TCGAbiolinks (version 2.30.4) in R (4.3.1). All other datasets supporting the current study are available from the corresponding author upon reasonable request. Statistical analysis Immuno-blot quantifications were performed using Image J software version 1.50i (NIH, Bethesda, MD). Statistical significance was calculated using Student’s t test in Graph Pad Prism 8. A value of p < 0.05 was considered statistically significant (* p < 0.05; ** p < 0.01; *** p < 0.001). Data are expressed as standard error of the mean (SEM) which are from at least three independent experiments. Results ULK1 Activity is Elevated in Pancreatic Cancer Independent of mRNA Expression To assess the clinical relevance of autophagy proteins in pancreatic cancer, we analyzed the expression of 25 core autophagy proteins in pancreatic adenocarcinoma (PAAD) from The Cancer Genome Atlas (TCGA). These autophagy gene sets were significantly upregulated in tumor tissues compared to adjacent normal tissues (P < 0.05; Supplementary Fig. S1A–C), and high expression levels of autophagy gene sets correlated with poor 5-year overall survival (Supplementary Fig. S1D), supporting the tumor-promoting role of autophagy in PDAC. Interestingly, the mRNA levels of ULK1 was not significantly different between tumor and normal tissues (Supplementary Fig. S1E). However, multiplex immunohistochemistry (multi-IHC) revealed elevated ULK1 protein levels and activity, marked by phosphorylated ATG14 (pATG14), particularly in high-grade (Grade 3) pancreatic tumors compared to normal tissues (Fig. 1A–D, Supplementary Fig. S2A, B). Similarly, increases in ULK1 and pATG14 levels were observed in human pancreatic cancer cell lines compared to normal human pancreatic epithelial (HPNE) and fibroblast (IMR90) cells (Fig. 1E). Moreover, elevated expression of ULK1 and pATG14 was detected in transformed pancreatic intraepithelial neoplasia (PanIN) in Kras +/LSL-G12D ; Pdx1-Cre (KC) mice and in advanced PDAC lesions from Kras +/LSL-G12D ; Trp53 R172H/+ ; Pdx1-Cre (KPC) mice (Fig. 1F). These results suggest that ULK1 activity rather than its mRNA levels is significantly elevated in malignant pancreatic cancers, supporting its potential role in tumor progression. ULK1 Depletion Suppresses Pancreatic Cancer Cell Proliferation, Invasion, and Autophagy To investigate the functional role of ULK1 in pancreatic cancer progression, we generated Ulk1 knockout (KO) KPC cells using CRISPR-Cas9, which was confirmed by genomic sequencing and qRT-PCR (Supplementary Fig. S3A, B). Ulk1 KO cells exhibited significantly impaired cell growth compared to control cells, as measured by IncuCyte live-cell imaging (Fig. 2A) and MTT assay (Fig. 2B). ULK1 loss also reduced colony formation (Fig. 2C) and markedly impaired 3D spheroid growth (Fig. 2D, E). Additionally, Transwell invasion assay showed a substantial decrease in invasion of Ulk1 KO cells compared to sgSC controls (Fig. 2F, G), indicating a significant reduction in both proliferative and invasive potential. To determine whether these phenotypes were linked to autophagy, we assessed autophagic flux by analyzing LC3 processing and localization. ULK1-deficient cells accumulated LC3-I with reduced conversion to LC3-II, particularly under amino acid-deprived conditions (Fig. 2H), and confirmed by a lower LC3-II/I ratio in Bafilomycin A1-treated cells (Supplementary Fig. S3C, D). Rapamycin-induced GFP-LC3 puncta formation was also significantly impaired in ULK1 knockdown cells (Fig. 2I, J) and, further confirming autophagy suppression and defect of viability (Supplementary Fig. S3E). These findings indicate that ULK1 is required for nutrient-responsive autophagy, and that its loss impairs both growth and invasion of pancreatic cancer cells. ULK1 Loss Delays Tumor Progression in Orthotopic Pancreatic Tumor Model To extend our in vitro findings, we investigated the in vivo role of ULK1 in tumor progression using syngeneic mouse model. An orthotopic PDAC mouse model was established by injecting Ulk1 KO (sg Ulk1 ) or control (sgSC) KPC cells into the pancreas of C57BL/6J mice. Histological analysis 3–5 weeks post-injection revealed reduced Ki67 staining in ULK1-deficient tumors compared to controls, consistent with decreased Ulk1 expression (Fig. 2K, L). Moreover, mice bearing Ulk1 KO tumor showed significantly extended survival compared to control KPC tumor-injecting mice (Fig. 2M). These findings support a critical role of Ulk1 in promoting cell proliferation and tumor progression in allograft in vivo model. Ulk1 Deficiency Suppresses Pancreatic Tumor Development in a Spontaneous Mouse Model To further investigate the role of ULK1 in pancreatic cancer progression, we utilized the LSL - Kras G12D/+ ; LSL - Trp53 R172H/+ ; Pdx1-Cre (KPC) mouse model (33), which spontaneously develops pancreatic tumors that mimic the pathology of human PDAC. These mice were crossbred with previously established Ulk1 fl/fl mice (24) to generate KPC;Ulk1 fl/fl mice, enabling pancreas-specific Ulk1 deletion via Cre recombinase (Fig. 3A). In this genetically engineered mouse (GEM) model, the pancreas-specific expression of Cre recombinase, driven by Pdx1 -Cre, induces a constitutively active Kras mutation (G12D) and a dominant negative Trp53 mutation (R172H) (Fig. 3A; Supplementary Fig. S4A). Immunoblotting confirmed efficient Cre-mediated Ulk1 deletion in pancreatic tissues (Fig. 3B). Histological analysis showed only significant abnormalities in pancreas rather than other tissues, though occasional lung pathology was observed in positive control ( KPC ; Ulk1 fl/+ ) mice (Fig. 3C) suggesting Ulk1 deletion specifically impacts pancreatic tumor development. However, pancreas weights were significantly reduced in KPC ; Ulk1 fl/fl mice compared to that of KPC control mice ( KPC ; Ulk1 fl/+ ) (Fig. 3D). Moreover, 18F-FDG-PET/CT imaging revealed a reduced tumor burden and lower SUVmax values in KPC ; Ulk1 fl/fl mice, compared to KPC ; Ulk1 fl/+ control mice (Fig. 3E; Supplementary Fig. S4B). Notably, increased FDG uptake in the lungs, indicative of distant metastasis, was observed in KPC ; Ulk1 fl/+ control mice but diminished in KPC ; Ulk1 fl/fl mice. These findings suggest that Ulk1 deletion primarily impacts pancreatic tumor development in the KPC model, with occasional lung metastasis in late stages of cancer. Next, further histopathological results from pancreas showed that all KPC control mice ( KPC ; Ulk1 fl/+ or KPC ; Ulk1 +/+ ) developed moderate-to-malignant PDAC within 18 weeks, whereas only 12 % (1/8) KPC ; Ulk1 fl/fl mice (pancreas-specific Ulk1 KO KPC) developed low -grade PanIN lesions, with the majority displaying normal pancreatic architecture with healthy acini cells (Fig. 4A, B). Immunohistochemistry (IHC) staining further revealed significantly lower levels for cytokeratin 19 (CK19) and Ki67 in KPC ; Ulk1 fl/fl pancreatic tissues compared to those from KPC control ( KPC ; Ulk1 fl/+ ) mice, consistent with suppressed epithelial transformation and proliferative capacity (Fig. 4C, D). These findings aligned with reduced expression of Ulk1 and pAtg14 in the pancreatic ductal regions of KPC ; Ulk1 fl/fl mice, which was more pronounced in the transformed regions of control KPC mice (Fig. 4C, D). In agreement with impaired autophagic flux, LC3B, an autophagy marker, was significantly decreased in the pancreas of KPC ; Ulk1 fl/fl (Supplementary Fig. S4C, D). Multiplex-IHC further confirmed that Ulk1 and pAtg14 levels were significantly enriched in Ck19 + epithelial cells within tumor from KPC ; Ulk1 fl/+ mice, while these signals were nearly absent in the tissue from KPC ; Ulk1 fl/fl mice (Fig. 4E, F). Importantly, KPC ; Ulk1 fl/fl mice exhibited a significantly prolonged median overall survival— approximately 10 weeks longer than that of KPC ; Ulk1 fl/+ controls (Fig. 4G). Spatial analysis supported a strong association between CK19⁺ adenocarcinoma cells in and pAtg14⁺ autophagy-active cells in KPC ; Ulk1 fl/+ tumors, which was largely diminished in Ulk1 deficient mice (Fig. 4H, I). Collectively, these data demonstrate that Ulk1 is critical for PDAC progression in a physiologically relevant spontaneous PDAC model and support its function as a key effector of both primary tumor growth and metastatic potential. Ulk1 Knockout Alters Tumor Immune Microenvironment and Enhances Antitumor Immunity Given the role of autophagy in modulating tumor immunity (37, 38), we next examined whether Ulk1 deletion alters immune cell composition within the tumor microenvironment (TME). Immunohistochemical analysis revealed that several TME markers increased tumor-associated fibroblasts (αSMA) and immunosuppressive M2 macrophages (CD204) in KPC ; Ulk1 fl/+ control mice tumor, particularly in CK19 + adenocarcinoma lesions, whereas these markers were significantly reduced in the pancreas of KPC ; Ulk1 fl/fl mice (Fig. 5A, B). Immunosuppressive immune cells neutrophils (Ly6G) were also stained stronger in KPC ; Ulk1 fl/+ control mice tumor rather than reduced in the pancreas of KPC ; Ulk1 fl/fl mice. In contrast, markers for tumor-infiltrating lymphocytes, such as cytotoxic T cells (CD8ɑ) and natural killer (NK) cells (NCR1), were more abundant in pancreas of Ulk1 KO mice ( KPC ; Ulk1 fl/fl ) than in control KPC mice ( Ulk1 wild-type (WT) or heterozygous KPC ( KPC ; Ulk1 fl/+ ), suggesting an enhanced antitumor immune response (Fig. 5A, B). Multiplex IHC further confirmed that, CD204 + M2 macrophages were more abundant in control tumor ( KPC ; Ulk1 fl/+ ) mice, while CD8 + T cells were more enriched in pancreas from KPC ; Ulk1 fl/fl mice than in control KPC mice, although statistical significance were not shown (Supplementary Fig. 5A–C). Taken together, these findings indicate that Ulk1 deletion remodels the pancreatic TME by reducing immunosuppressive immune cells and increasing cytotoxic lymphocyte infiltration. ULK1 Deletion Reshapes Immune Cell Composition in Pancreatic Tumors To elucidate the molecular mechanisms underlying tumor regression following Ulk1 depletion, we performed RNA-sequencing analysis of Ulk1 WT and Ulk1 KO KPC cells. Differential expression analysis identified 4,097 genes with altered expression including 1071 upregulated and 1538 downregulated in Ulk1 KO cells. KEGG pathway enrichment analysis of upregulated gene sets in Ulk1 KO cells revealed significant enrichment “MAPK signaling”, “PI3K-Akt signaling” and “Cytokine-cytokine receptor interaction” pathways (Supplementary Table S3). Proteomic profiling of KRas G12D -expressing HPNE cells treated with chloroquine (CQ) revealed a similar upregulation of immune-related pathways, with “Cytokine–cytokine receptor interaction”, “JAK–STAT signaling”, “TGF-β signaling”, and “Mitophagy” among the most enriched pathways (Supplementary Table S4). Complementing these results, gene set enrichment analysis (GSEA) of TCGA pancreatic cancer datasets (34) revealed that lower ULK1 expression correlated with elevated immune and mitochondrial gene signatures. Gene ontology (GO) analysis of gene clusters negatively co-expressed with ULK1 revealed strong enrichment for terms related to “Antigen processing and presentation”, "Immune system processes", and “Mitochondrial metabolism”, suggesting a link between ULK1 suppression and activation of immune and mitochondria metabolic pathways (Supplementary Table S5). To further assess whether tumor-intrinsic Ulk1 deletion affects immune cell composition in vivo , we employed syngeneic orthotopic models by injecting KPC sgSC ( Ulk1 WT) and KPC sg Ulk1 ( Ulk1 KO) cells into the pancreas of immunocompetent C57BL/6J mice. After 3 weeks, the mice were sacrificed and subjected to immunophenotyping by FACS analysis using the gating strategy shown in Supplementary Fig. S6. Although the overall proportions of tumor-infiltrating CD45 + immune cells and CD45 + CD11b + myeloid cells remained unchanged between the two groups, Ulk1 KO-derived tumor showed a marked reduction in the proportion of neutrophils (CD45 + CD11b + Ly6G + ) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs; CD11b + Ly6G + F4/80 − ) compared with tumors derived from Ulk1 WT KPC cells, alongside a significant increase in MHC class II + antigen-presenting cells (APCs) (CD45 + CD11b + MHC-II + ) in tumor derived from Ulk1 KO KPC cells (Fig. 6A, B). In the lymphoid compartment, Ulk1 KO cell-derived tumors exhibited higher proportion of CD8 + T cells over CD4 + T cell ratio than in Ulk1 WT tumors, though total CD3 + or CD4 + T cell proportions were unchanged (Supplementary Fig. S6B, S7A). This increase in cytotoxic T cells may be linked to enhanced recruitment of MHC class II + APCs in Ulk1 KO tumors. To validate these findings in a spontaneous PDAC model, we performed similar immunophenotyping in KPC control and KPC;Ulk1 fl/fl mice (Fig. 6C, D). Similar to the orthotopic model, we observed increased proportions of MHC II + APC cells, and significantly reduced neutrophils and PMN- in KPC;Ulk1 fl/fl mice. In lymphoid populations, CD8 + T cell proportions were considerably elevated in KPC;Ulk1 fl/fl mice, whereas the CD4 + T cell proportion remained comparable to that in KPC;Ulk1 fl/+ control mice, consistent with the results of the syngeneic orthotopic model (Supplementary Fig. S7B). Collectively, these data from two independent in vivo models indicate that tissue-specific Ulk1 depletion enhances antitumor immune responses by markedly reducing immuno-suppressive PMN-MDSCs and neutrophils while promoting APCs and CD8 + T cell infiltration, thus contributing to tumor regression. Ulk1 regulates Cytokines and Chemokine Secretion to Support a Pro-Tumorigenic Immune Microenvironment To further dissect how ULK1 shapes the immune landscape in pancreatic tumors, we profiled cytokine and chemokine expression in pancreatic tissues from KPC control ( KPC ; Ulk1 fl/+ ) and KPC Ulk1 KO ( KPC ; Ulk1 fl/fl ) mice. Several pro-tumorigenic cytokines and chemokines including ICAM-1/CD54, TIMP1, IL-1RN (IL-1ra), and IL-16 (34, 39-41) were upregulated in KPC control pancreatic tissues compared with those in KPC ; Ulk1 fl/fl mice (Supplementary Fig. 8A, B). While Ccl2 was modestly downregulated in KPC;Ulk1 fl/fl tissues, Cxcl12 levels remained unchanged. Cytokine and chemokine profiling of conditioned media (CM) from Ulk1 WT and Ulk1 KO KPC cells revealed distinct secretory profiles, reinforcing the tumor-promoting immune landscapes (Fig. 7A, B). While Icam-1/CD54 was remained undetectable, and Timp1 levels were comparable in CM from KPC sgSC and KPC sg Ulk1 cells, suggesting these chemokines originate from fibroblasts rather than from tumor epithelial and immune cells. Pro-tumorigenic chemokines Cxcl2 and Ccl2, which promoting recruitment of PMN-MDSCs and M2 macrophages (42), were substantially reduced in Ulk1 KO cells, consistent with previous immunophenotyping data from pancreatic tissues from spontaneous cancer model (Fig. 6). Additionally, the secreted levels of G-CSF and Ccl2 were notably reduced, whereas GM-CSF levels were markedly elevated in in CM from Ulk1 KO cells, indicating that ULK1 determines to regulate tumor-intrinsic G-CSF, Ccl2 and GM-CSF secretion oppositely (Fig. 7A, B). These findings were confirmed by qPCR, which reduced expression of Ccl2 , IL-1rn , Cxcl2 , and Csf3 (G-CSF) in Ulk1 KO cells but increased Csf2 (GM-CSF) expression (Fig. 7C). ELISA results also exhibited the concentration of Ccl2, G-CSF and GM-CSF secreted from both genotyped cells, which were consistent with their gene expression levels (Fig. 7D). Together, these data demonstrate that Ulk1 depletion suppresses secretion of key cytokines and chemokines that support the recruitment of tumor-promoting immune cell subsets. However, Ulk1 loss oppositely increased a crucial cytokine such as GM-CSF that promotes the tumor-suppressive immune cell populations, through tumor-intrinsic signaling, ultimately leading to a less immune-suppressive TME and further reduced tumor progression. ULK1-Dependent Cytokine Signaling Influences Immune Cell Survival To investigate whether tumor-derived factors directly influence tumor-infiltrated immune cell survival, we isolated freshly immune cells from either pancreas tumor tissues or blood/spleen of tumor-bearing KPC mice at 15 weeks old and sorted neutrophils including PMN-MDSC (CD45 + CD11b + Ly6G + ), cytotoxic T cells (CD45 + CD3 + CD8 + ) and dendritic cells (DCs; CD45 + CD11c + ), respectively. These sorted immune cells were cultured in CM from either Ulk1 -WT or KO KPC cells for 24 h and then assessed their viability by Calcein-AM staining. Neutrophils including PMN-MDSCs from tumors showed significantly higher viability cultured in CM from KPC WT cells (Fig. 7E; Supplement Fig. S9A). In contrast, the viability of CD8 + T cells and dendritic cells (DC) from tumor was significantly higher in Ulk1 KO CM than in KPC WT (Fig. 7 E; Supplement Fig. S9A). Interestingly, these viability differences were not observed in immune cells isolated from blood or spleen of tumor-bearing KPC mice when cultured in the same CM, suggesting that Ulk1-dependent tumor -secreted factors specifically affect immune cells within the tumor context (Supplement Fig. S9B, C). Collectively, these results reveal that ULK1 orchestrates a tumor-intrinsic cytokine/chemokine program that shapes an immunosuppressive microenvironment by supporting the survival of neutrophils including PMN-MDSCs and suppressing DC and cytotoxic T cell function—mechanisms which are disrupted upon ULK1 deletion. Finally, to evaluate the translational relevance of these findings, we analyzed tissue microarrays (TMAs) from human PDAC samples. Grade 3 adenocarcinoma regions displayed higher CD163 + M2 macrophage staining, while the infiltration of CD8 + cytotoxic T cells and MHC II + APCs was lower in tumor lesions (Adeno) than in adjacent normal tissue (CTL) (Fig. 7F, G). This staining pattern was also observed in TMA with grade 1 and 2 (Supplementary Fig. S10A, B), consistent with high ULK1 activity observed in human pancreatic cancers (Fig. 1A and Supplementary Fig. S2A). Taken together, these findings indicate that Ulk1 deletion remodels the tumor immune microenvironment by reducing tumor-promoting myeloid cell such as M2 macrophages and neutrophils/PMN-MDSCs, and increasing cytotoxic T cells and APCs, leading to enhanced antitumor immunity and suppressed tumor growth. Discussion Autophagy plays a context-dependent role in cancer, functioning as a tumor suppressor during early tumorigenesis but promoting tumor survival in established malignancies. In PDAC, elevated autophagy activity supports tumor progression by providing essential metabolic substrates for cancer cell survival. Although the importance of autophagy in PDAC has been demonstrated by the tumor-suppressive effects of Atg5 and Atg7 deletion (4, 5), the role of Unc-51-like kinase 1 (ULK1), a key initiator of autophagy, remains underexplored in in this context. Here, we suggest ULK1 as a critical regulator of pancreatic tumor progression through both cell-intrinsic and immune-modulatory mechanisms. Our findings support a previously unrecognized role for ULK1 in generating a pro-tumorigenic immune microenvironment and suggest that targeting ULK1 may offer a dual therapeutic benefit by impairing autophagy and promoting antitumor immunity. Although ULK1 mRNA expression is unchanged between normal and tumor patients from human PDAC datasets (TCGA) (Supplementary Fig. S1), our multiplex-IHC and immunoblot analysis revealed that elevated ULK1 activity, as marked by phospho-Atg14 levels (pAtg14), in high-grade human PDAC tissues and cells (Fig. 1), suggesting that ULK1 activity, rather than its expression, is important for tumor progression. The functional significance of ULK1 in tumor progression is further supported by our findings that ULK1 depletion in both mouse (KPC) and human (MIA PaCa-2) pancreatic cancer cells significantly impaired their proliferation and invasion, supporting its role in tumor aggressiveness (Fig. 2 and Supplementary Fig. S3). Consistent with its crucial role in autophagy activation, ULK1-depleted cells showed defective autophagosome formation, as indicated by reduced LC3-II/I ratios and GFP-LC3 puncta under stress conditions, confirming that ULK1 is critical for maintaining autophagy flux and metabolic adaptation for cancer growth (Fig. 2 and Supplementary Fig. S3). Importantly, our in vivo studies using both syngeneic orthotopic allografts and spontaneous KPC GEM models provide compelling evidence that Ulk1 is required for pancreatic tumor development in physiological setting. Tumor-intrinsic deletion of Ulk1 significantly delayed tumor onset, reduced PDAC burden, and extended survival (Fig. 2, 3 and 4). This study is the first direct evidence to demonstrate tumor-promoting function of Ulk1 in a spontaneous PDAC model, suggesting its potential as a promising therapeutic target. Beyond its canonical role in autophagy-dependent tumor survival, we found an immune-modulatory function of ULK1 in the PDAC tumor microenvironment (TME). Analysis of TCGA datasets revealed a negative correlation between ULK1 expression and MHC class II-mediated antigen presentation pathways (Supplementary Table S5), implicating that ULK1 may suppress antitumor immunity by inhibiting antigen processing in tumor and suppressing antigen presenting cells (APCs) (34, 37). While autophagy has been implicated in immune evasion, particularly through MHC-I degradation of tumor cells (37), our data further revealed that Ulk1 -deficient tumors markedly increased the infiltration of MHC II + antigen presenting cells (APCs) into tumors, likely potentially enhancing tumor antigen presentation and T cell priming (Fig 5 and Supplementary Fig S5-7). Moreover, genetic deletion of Ulk1 in tumor significantly altered the composition of the TME across both orthotopic and spontaneous KPC models. We observed a marked reduction in immunosuppressive polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and neutrophils, accompanied by an increase in CD8⁺ cytotoxic T cells and MHC II⁺ APCs (Fig. 5, 6, and Supplementary Fig. S5-S7). These consistent observations across models strongly suggest that ULK1 contributes to immune evasion by orchestrating the recruitment and maintenance of suppressive myeloid populations while restricting cytotoxic immune cell infiltration. Mechanistically, cytokine and chemokine profiling revealed that Ulk1-deficient tumors secreted lower levels of Ccl2, Cxcl2, and G-CSF, key factors that recruit immunosuppressive myeloid cells (43-52). Interestingly, among the cytokines, GM-CSF was the most prominently elevated in Ulk1 KO tumors (Fig. 7). Although dual roles of GM-CSF in TME by promoting or inhibiting immune cell subtypes have been reported (53-55), recently GM-CSF shows anti-tumor immune responses by activating M1-macrophages and enhancing DCs differentiation (56-58). The upregulation of DCs populations may explain the increased viability of APCs and further activated T cells observed in Ulk1 -deficient tumors (Fig. 5, 6, Supplementary Fig. S5 and S6). These tumor-intrinsic cytokine changes were recapitulated in vitro using conditioned media (CM) from Ulk1 KO KPC cells, confirming that Ulk1 -mediated tumor signaling influences immune cell recruitment through determined by the types of cytokines and chemokines (Fig. 7A-D). Functional assays further demonstrated that conditioned media from Ulk1 KO cells impaired the survival of tumor-infiltrating neutrophils including PMN-MDSCs, while enhancing the viability of CD8⁺ T cells and DCs (Fig. 7E-G and Supplementary Fig. S9A-C). These results suggest that Ulk1-dependent cytokine secretion actively dictates immune cell fate within the tumor niche. These findings highlight the role of Ulk1 in maintaining an immunosuppressive TME, promoting tumor growth not only through autophagy-driven metabolic support but also by modulating immune cell compositions and dynamics. Previous studies have shown that autophagy promotes the secretion of pro-tumorigenic factors—including IL-6, IL-8, and MMP2—in Ras-driven cancers and facilitates IL-1β secretion through unconventional secretory pathways (59). In line with these studies, our data indicate that ULK1 plays an important role in regulating the secretion of cytokine and chemokines that reprogram the tumor immune landscape. While the precise molecular mechanisms by which ULK1 regulates cytokine expression and trafficking remain to be fully elucidated, our findings strongly implicate ULK1-mediated tumor signals as acting a crucial role in coordinating tumor–immune interactions through selective modulation of immune-regulatory chemokines and cytokines. In summary, our study suggests ULK1 as a critical regulator of pancreatic cancer progression through dual mechanisms: sustaining tumor metabolism via autophagy and fostering an immunosuppressive TME by modulating cytokine-mediated immune cell recruitment and survival. These findings support the therapeutic potential of targeting ULK1 in PDAC, offering a strategy to simultaneously impair tumor-intrinsic metabolic support and enhance antitumor immunity. Declarations ACKNOWLEDGEMENT We thank T. Kim (Flow Cytometry Core), K. Kim (Proteomics Core), M. Kim (Microscopy Core), S. Jeon (Molecular Imaging Core), and M. Park (Laboratory Animal Research Core) from Core Center, NCC Korea. The animal study protocols were approved by the Institutional Animal Care and Use Committee of National Cancer Center (Approval #NCC-22-815; # NCC-24-1045) and conducted in accordance with ARRIVE guidelines. This work was supported by the National Cancer Center (NCC-24H1200 and 2510750) and National Research Foundation of Korea (2020R1A2B5B01002011). AUTHORS ' DISCLOSURES No disclosures were reported. AUTHORS CONTRIBUTION H. Jeong: Conceptualization, formal analysis, validation, investigation, visualization, methodology, writing–original draft. J. Lee: Conceptualization, formal analysis, validation, investigation, visualization, methodology, writing–original draft. J. Son: Conceptualization, formal analysis, data curation, investigation, visualization, methodology. J. Lee: Investigation, methodology. M. Kang: Investigation, methodology. S. Cho: Methodology. J.H. Kim: Methodology. Y. 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Additional Declarations There is no conflict of interest Supplementary Files SupplementaryInfov11EMMCheong.docx Supplementary Info Cite Share Download PDF Status: Published Journal Publication published 17 Dec, 2025 Read the published version in Experimental & Molecular Medicine → Version 1 posted Editorial decision: revise 03 Jun, 2025 Review # 1 received at journal 01 Jun, 2025 Review # 2 received at journal 25 May, 2025 Reviewer # 2 agreed at journal 13 May, 2025 Reviewer # 1 agreed at journal 13 May, 2025 Reviewers invited by journal 08 May, 2025 Submission checks completed at journal 13 Apr, 2025 Editor assigned by journal 13 Apr, 2025 First submitted to journal 13 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Korea","correspondingAuthor":false,"prefix":"","firstName":"Miju","middleName":"","lastName":"Kang","suffix":""},{"id":454018036,"identity":"53d0bb34-88d0-4405-965d-5587d70d8d5a","order_by":6,"name":"Sunghyeon Cho","email":"","orcid":"","institution":"National Cancer Center Korea","correspondingAuthor":false,"prefix":"","firstName":"Sunghyeon","middleName":"","lastName":"Cho","suffix":""},{"id":454018037,"identity":"132cf675-d987-4775-89f5-b31140c1f41d","order_by":7,"name":"Ji Hyeon Kim","email":"","orcid":"","institution":"National Cancer Center Korea","correspondingAuthor":false,"prefix":"","firstName":"Ji","middleName":"Hyeon","lastName":"Kim","suffix":""},{"id":454018038,"identity":"9dfdf1ae-532c-455f-b1f6-bef5949407c0","order_by":8,"name":"Yoon Jeon","email":"","orcid":"","institution":"National Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Yoon","middleName":"","lastName":"Jeon","suffix":""},{"id":454018039,"identity":"4010b5ec-ae85-4b3e-b4c8-1ad636eea170","order_by":9,"name":"Jonghyun Lee","email":"","orcid":"","institution":"National Cancer Center Korea","correspondingAuthor":false,"prefix":"","firstName":"Jonghyun","middleName":"","lastName":"Lee","suffix":""},{"id":454018040,"identity":"7c44f3c1-97c9-425d-87b1-712dc522ad7c","order_by":10,"name":"Dongkwan Shin","email":"","orcid":"https://orcid.org/0000-0002-6925-1081","institution":"National Cancer Center Korea","correspondingAuthor":false,"prefix":"","firstName":"Dongkwan","middleName":"","lastName":"Shin","suffix":""},{"id":454018041,"identity":"0e972615-7ad2-4ba3-9f7e-dcbf9b4cf156","order_by":11,"name":"Hye-Ran Kim","email":"","orcid":"","institution":"National Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Hye-Ran","middleName":"","lastName":"Kim","suffix":""},{"id":454018042,"identity":"240dc824-3ee3-45d0-b884-4969f76983ff","order_by":12,"name":"Ho Lee","email":"","orcid":"https://orcid.org/0000-0001-5573-742X","institution":"National Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Ho","middleName":"","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2025-04-13 09:55:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6438501/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6438501/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s12276-025-01590-2","type":"published","date":"2025-12-17T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82637271,"identity":"0a63be5e-c2b6-4f35-814d-b3c76883290f","added_by":"auto","created_at":"2025-05-13 14:41:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":117536126,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eULK1 expression and activities are upregulated in pancreatic cancers.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B) \u003c/strong\u003eRepresentative images (A) and quantitative data (B) of ULK1 (Yellow) and pATG14 (Red) in human pancreatic adenocarcinoma (PAAD) tissue microarray (TMA) using multiplex IHC. Tissues from six patients were analyzed, each including matched adjacent normal and tumor tissue (four cores per patient). Images fromrepresentative Grade 3 PAAD cases areshown in this figure. C7/C8 cores indicate adjacent normal pancreas tissue (CTL) while C5/C6 represent adenocarcinoma (Adeno) tumor regions. Scale bars are indicated in the pictures. Error bars indicate the mean ± SEM from two independent cores (ULK1 p=0.2295; pATG14 p=0.0050). \u003cstrong\u003e(C, D) \u003c/strong\u003eRepresentative images (C) and quantification (D) of pATG14 by grade using IHC staining in adenocarcinoma cores from human PAAD TMA, stratified by tumor grade (Grade 1 vs 3 p=0.0016). \u003cstrong\u003e(E)\u003c/strong\u003eImmunoblot analysis of ULK1, pATG14, ATG101 and β-Actin (loading control) in human pancreatic cancer cell lines. \u003cstrong\u003e(F)\u003c/strong\u003eRepresentative images of H\u0026amp;E staining and IHC staining for CK19, Ulk1 and pAtg14 in pancreas tissue from K, KC and KPC mice. Scale bars are indicated in the pictures. All values were considered statistically significant by Student’s t-test (*p\u0026lt;0.05; **p\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6438501/v1/4d2246f039a4c54a663bb4f6.png"},{"id":82637985,"identity":"07c7dec8-bedc-4cdc-a890-9bc9d173806a","added_by":"auto","created_at":"2025-05-13 14:49:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110714343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUlk1 regulates tumorigenesis in an orthotopic pancreatic cancer model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eRelative cell proliferation between sgScrambled (sgSC), sg\u003cem\u003eUlk1\u003c/em\u003e KPC clone 1(c1) and clone 2(c2) cells analyzed by IncuCyte® over 48h (p \u0026lt; 0.0001 for all time points after 2 h). \u003cstrong\u003e(B) \u003c/strong\u003eRelative cell viability between sgSC, sg\u003cem\u003eUlk1\u003c/em\u003e c1 and c2 KPCcells analyzed by MTT assay over 72h (sgSC vs. sg\u003cem\u003eUlk1\u003c/em\u003e c1 p=0.0015, sgSC vs. sg\u003cem\u003eUlk1\u003c/em\u003e c2 p=0.0022). \u003cstrong\u003e(C) \u003c/strong\u003eThe colony formation assays using sgSC, sg\u003cem\u003eUlk1\u003c/em\u003e c1 and c2 KPC cells. \u003cstrong\u003e(D, E) \u003c/strong\u003eBright field images (D) and quantification (E) of showing 3D spheroid formation using sgSC, sg\u003cem\u003eUlk1\u003c/em\u003e c1 and c2 KPC cells. Image analysis and quantification were performed using the ImageJ program (sgSC vs. sg\u003cem\u003eUlk1\u003c/em\u003ec1 p=0.1049, sgSC vs. sg\u003cem\u003eUlk1\u003c/em\u003e c2 p=0.1705). \u003cstrong\u003e(F, G) \u003c/strong\u003eRepresentative images captured at 4x and 20x magnification (F) and quantification (G) of Transwell migration and Matrigel invasion assays analyzed using sgSC, sg\u003cem\u003eUlk1\u003c/em\u003e c1 and c2 KPC cells (sgSC vs. sg\u003cem\u003eUlk1\u003c/em\u003e c1 p=0.0001, sgSC vs. sg\u003cem\u003eUlk1\u003c/em\u003e c2 p=0.0001). \u003cstrong\u003e(H) \u003c/strong\u003eImmunoblot analysis of Ulk1, pAtg14, Atg14, LC3 and β-Actin (loading control) in sgSC or sg\u003cem\u003eUlk1 \u003c/em\u003eKPC cells cultured in complete medium (COM) or replaced by amino acid-deprived (-A.A) media for 2 hours. \u003cstrong\u003e(I, J) \u003c/strong\u003eRepresentative images of GFP-LC3 puncta (I) and quantification (J) in MIA PaCa-2 cell lineharboring shControl (shCTL) or sh\u003cem\u003eULK1\u003c/em\u003eafter treatment with either 4µM of rapamycin (Rapa) \u0026nbsp;or vehicle (Veh) for 4 hours. \u003cstrong\u003e(K, L) \u003c/strong\u003eRepresentative images (K) and quantitative data (L) of H\u0026amp;E staining and Ki67 and Ulk1 by IHC in pancreatic tissues obtained from orthotopic models of PDAC injected by KPC sgSC or KPC sg\u003cem\u003eUlk1\u003c/em\u003e (Ki67 p=0.0008/H-score p=0.5206, Ulk1 p\u0026lt;0.0001/H-score p\u0026lt;0.0001). \u003cstrong\u003e(M) \u003c/strong\u003eSurvival graph illustrating orthotopic mouse models of PDAC, with each group consisting of n=5 mice (p=0.0214). The models were generated using KPC cells carrying either sgSC or sg\u003cem\u003eUlk1\u003c/em\u003e, and the observation period extended up to 119 days. Scale bars are indicated in the pictures and error bars indicate the mean ± SEM for three independent images. All values were considered statistically significant by Student’s t-test (*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6438501/v1/5bec915ab65ae9b7a994fb09.png"},{"id":82637268,"identity":"0508dd80-75cc-487b-aef4-a3aff7b28338","added_by":"auto","created_at":"2025-05-13 14:41:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":62106851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePancreas-specific \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eUlk1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deletion suppresses pancreatic cancer development in KPC GEM model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Breeding strategy for generation of \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice. \u003cstrong\u003e(B)\u003c/strong\u003e Immunoblot analysis of ULK1, pATG14 and β-Actin (loading control) in indicated organs of \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mouse compare with controls (\u003cem\u003eC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e). \u003cstrong\u003e(C)\u003c/strong\u003e Pancreas morphology phenotypes (top) and H\u0026amp;E staining (bottom) of various organs from normal control mouse (CTL), \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e, and \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice. \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of pancreas weight (n=6; p=0.0207). Error bars indicate the mean ± SEM. \u003cstrong\u003e(E)\u003c/strong\u003e PET-CT imaging showing tumors indicated by arrows from CTL, \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6438501/v1/1cb74d1fc379491900d11ede.png"},{"id":82637981,"identity":"53981309-1949-4ce3-9b87-7a5eea28b927","added_by":"auto","created_at":"2025-05-13 14:49:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1946638,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUlk1 activity is associated with pancreatic tumor transformation in KPC mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B)\u003c/strong\u003e Representative images of H\u0026amp;E staining (A) and quantification of tumor area (B) in pancreatic tissues from \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+ \u003c/em\u003e\u003c/sup\u003eand \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e GEM mice (n=6; p=0.0087). Error bars indicate the mean± SEM for six independent experiments (p=0.0087). \u003cstrong\u003e(C, D)\u003c/strong\u003e Representative images (C) and quantitative data (D) of H\u0026amp;E staining and CK19, Ki67, Ulk1 and pAtg14 by IHC in pancreas tissues from \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+ \u003c/em\u003e\u003c/sup\u003eand \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice (CK19 H-score p=0.017, Ki67 H-score p=0.0035, Ulk1 H-score p=0.0386, and pAtg14 H-score p=0.0187). \u003cstrong\u003e(E, F) \u003c/strong\u003eMultiplex IHC images (E) and quantification (F) for H\u0026amp;E, CK19 (White), Ulk1 (Yellow) and pAtg14 (Red) in pancreas tissues from \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+ \u003c/em\u003e\u003c/sup\u003eand \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice (CK19 p=0.0898, Ulk1 p=0.5825, pATG14 p=0.0255). Scale bars are indicated in the pictures and error bars indicate the mean± SEM for over four independent experiments. \u003cstrong\u003e(G)\u003c/strong\u003e Kaplan-Meier survival analysis of \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+ \u003c/em\u003e\u003c/sup\u003e(\u003cem\u003en\u003c/em\u003e=10) and \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003e(\u003cem\u003en\u003c/em\u003e=9) mice (p=0.0011). \u003cstrong\u003e(H, I)\u003c/strong\u003e Spatial analysis of CK19\u003csup\u003e+\u003c/sup\u003e and pAtg14\u003csup\u003e+\u003c/sup\u003e cells with quantification of their proximity in tumor tissues. Heat map showing median distance (H left) and cell count (H right) and quantitative data (I) representing pAtg14+ cells near or far from CK19+ malignant cell phenotypes across the mouse cohort. Error bars indicate the mean± SEM for three independent experiments. (at 10µm: p=0.1313). All values were considered statistically significant by Student’s t-test (*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6438501/v1/93c3b0e36129ef2462f27c35.png"},{"id":82637257,"identity":"008257ba-7c60-474e-a371-e41f66cbee63","added_by":"auto","created_at":"2025-05-13 14:41:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16010511,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePancreas-specific \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eUlk1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e depletion alters tumor immune microenvironment in KPC mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B) \u003c/strong\u003eRepresentative images (A) and quantitative data (B) of H\u0026amp;E staining and αSMA, CD8α, CD204, Ly6G and NCR1 in pancreas tissue from \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice. Scale bars are indicated in the pictures and error bars indicate the mean± SEM for over four independent experiments (αSMA p=0.0086, CD8α p=0.2718, CD204 p=0.0350, Ly6G p=0.0121, NCR1 p=0.1920). All values were considered statistically significant by Student’s t-test (*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6438501/v1/60771c0d4856ead5a2d221fe.png"},{"id":82637274,"identity":"c4da55a9-c389-4b96-a92f-e782dbf64a94","added_by":"auto","created_at":"2025-05-13 14:41:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":57931678,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTumor Ulk1 deletion modulates myeloid cell recruitment in both orthotopic and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKPC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e GEM model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B)\u003c/strong\u003e Representative flow cytometry plots (A) and quantification (B) of the expression by the myeloid immune cell types analyzed by FACS in pancreas tissue from the orthotopic mouse model (n \u0026gt; 3; CD45\u003csup\u003e+\u003c/sup\u003e p=0.376302, Myeloid cell p=0.742007, MHCII\u003csup\u003e+\u003c/sup\u003e p=0.003583, PMN-MDSC p=0.000378, Neutrophil p=0.000079). The 7-week-old female C57BL/6 mice were injected with KPC sgSC or KPC sg\u003cem\u003eUlk1\u003c/em\u003e cells and then sacrificed 3 weeks later. \u003cstrong\u003e(C, D)\u003c/strong\u003e Representative flow cytometry plots (C) and quantification (D) of the expression by the myeloid immune cell types analyzed by FACS in pancreas tissue from the 12 ~ 14-week-old \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e GEM mice (n\u0026gt; 4; CD45\u003csup\u003e+\u003c/sup\u003e p=0.378793, Myeloid cell p=0.038968, MHCII\u003csup\u003e+\u003c/sup\u003e p=0.391091, PMN-MDSC p=0.007721, Neutrophil p=0.005358). Error bars indicate the mean± SEM.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6438501/v1/d7d7c463579b6130f2b1be02.png"},{"id":82637982,"identity":"e7599ec3-dd8d-4f49-8f43-c5389614a435","added_by":"auto","created_at":"2025-05-13 14:49:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":37018877,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUlk1 regulates the expression and secretion of cytokines and chemokines critical to the tumor microenvironment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B)\u003c/strong\u003e Cytokine array images (A) and quantification (B) of conditioned media from sgSC or sg\u003cem\u003eUlk1\u003c/em\u003e KPC cells. Key differences were observed in G-CSF (p = 0.0002), GM-CSF (p=0.0040) IL-1ra (p = 0.0048), Ccl2 (p = 0.0022), Cxcl2 (p = 0.0104), and Timp1 (p = 0.0064). Red squares indicate cytokines with notable differences between two groups and bar graphs mean pixel densities analyzed by ImageJ. \u003cstrong\u003e(C)\u003c/strong\u003e qPCR analysis of cytokines and chemokines mRNA in sgSC or sg\u003cem\u003eUlk1\u003c/em\u003e KPC cells. (\u003cem\u003eCcl2\u003c/em\u003e p\u0026lt;0.0001, \u003cem\u003eIl1ra\u003c/em\u003e p=0.539; F, \u003cem\u003eCxcl1\u003c/em\u003e p=0.0142, \u003cem\u003eCxcl2\u003c/em\u003e p=0.0201, \u003cem\u003eCxcl12\u003c/em\u003e p=0.7400, \u003cem\u003eCsf3\u003c/em\u003e p=0.0258). \u003cstrong\u003e(D)\u003c/strong\u003e The secreted levels of cytokines from sgSC or sg\u003cem\u003eUlk1\u003c/em\u003e KPC cells using ELISA analysis (G-CSF p=0.0017, GM-CSF p\u0026lt;0.0001, Ccl2 p=0.0362). Each type of cells was seeded, incubated for 24 h, and then harvested for either RNA extraction of cells or conditioned media. Error bars indicate the mean ± SEM for over three independent experiments. \u003cstrong\u003e(E)\u003c/strong\u003e Viability analysis of sorted immune cells cultured with conditioned media from sgSC or sg\u003cem\u003eUlk1\u003c/em\u003e cells. Neutrophil including PMN-MDSCs(CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e cells), Cytotoxic T cells(CD45\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e cells), and DCs (CD45\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e cells) from pancreas in 15 ~ 16-week-old \u003cem\u003eKPC;Ulk1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/fl\u003c/em\u003e\u003c/sup\u003e mice were sorted, and then cultured with conditioned media from KPC sgSC or KPC sg\u003cem\u003eUlk1\u003c/em\u003e (sgSC CM or sg\u003cem\u003eUlk1\u003c/em\u003e CM) for 24 h, which were measured Calcein-stained live cells by Operetta CLS. Scale bars are indicated in the pictures and error bars indicate the mean ± SEM for over two independent wells (PMN-MDSCs + Neutrophils in pancreas Count p=0.0668/Normalized p=0.0124, Cytotoxic T cells in pancreas Count p=0.6024/Normalized p=0.3923, DCs in pancreas Count p=0.0637/Normalized p=0.0298).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F, G)\u003c/strong\u003e Representative images (F) and quantitative data (G) of CD163 (Orange), CD8α (Cyan) and MHCII (Magenta) by multiplex IHC in Grade 3 human pancreatic adenocarcinoma (PAAD) TMA. C7/C8 cores represent cancer adjacent pancreas tissue or adjacent normal pancreas tissue (CTL) but C5/C6 represent adenocarcinoma (Adeno). Scale bars are indicated in the pictures and error bars indicate the mean ± SEM for two independent cores (CD163 p=0.0453, CD8α p=0.0004, MHCII p=0.0115). All values were considered statistically significant by Student’s t-test (*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; ****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6438501/v1/b5e58eb57eed770a01279c80.png"},{"id":82637259,"identity":"aed656ea-b093-4ba0-b722-b2d62c453206","added_by":"auto","created_at":"2025-05-13 14:41:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7004023,"visible":true,"origin":"","legend":"Supplementary Info","description":"","filename":"SupplementaryInfov11EMMCheong.docx","url":"https://assets-eu.researchsquare.com/files/rs-6438501/v1/051ca41f1c9f71bf859ff894.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"ULK1 Knockout suppresses Pancreatic Cancer Progression by Inhibiting Autophagy and Enhancing Anti-tumor Immunity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAutophagy is a conserved intracellular degradation pathway that sustains cellular homeostasis by recycling macromolecules and clearing damaged organelles. Autophagy plays a crucial role in balancing normal cell functions and multiple pathophysiological conditions, including cancer. While it preserves genome stability in early cancer, it later supports tumor survival by maintaining organelles, enabling recycling, and fulfilling metabolic demands (1, 2).\u003c/p\u003e\n\u003cp\u003eThe tumor-promoting role of autophagy has been validated in genetically engineered mouse (GEM) models. Oncogenic mutations in \u003cem\u003eHRas\u0026nbsp;\u003c/em\u003eor \u003cem\u003eKRas\u003c/em\u003e increase basal autophagy, which is necessary for tumor cell survival during tumorigenesis (3). Deletion of \u003cem\u003eAtg5\u003c/em\u003e or \u003cem\u003eAtg7\u003c/em\u003e in KRas\u003cem\u003e\u003csup\u003eG12D\u003c/sup\u003e\u003c/em\u003e-driven spontaneous lung cancer models reduced tumor size compared with that in wild-type (WT) mice (4, 5). Melanomas with BRAF mutations (6) and pancreatic cancers harboring 95%\u0026nbsp;\u003cem\u003eKRas\u0026nbsp;\u003c/em\u003emutations also exhibit\u0026nbsp;high\u0026nbsp;dependency on autophagy genes\u0026nbsp;such as\u0026nbsp;\u003cem\u003eAtg5\u003c/em\u003e and \u003cem\u003eAtg7\u0026nbsp;\u003c/em\u003e(7, 8).\u0026nbsp;In addition,\u0026nbsp;depleting Atg proteins like FIP200, Atg16, or Atg4 suppresses tumor growth in various cancers\u0026nbsp;(9-11). Accordingly, autophagy inhibition has been considered a promising therapeutic strategy\u0026nbsp;(7, 8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUnc-51-like kinase 1 (ULK1), a serine/threonine kinase, has gained attention as a druggable target, which plays a crucial role in the initiation step of autophagy (12). ULK1 forms a complex with proteins such as ATG13, ATG101, and FIP200/RB1CC1, facilitated by the phosphorylation of ATG13 (13-15), and subsequently phosphorylating the components of the Class III PI(3) Kinase/Vps34 complex, including ATG14 and Beclin1 to drive autophagosome formation (16-18). ULK1 activity is tightly controlled by upstream nutrient-signaling molecules such as mTORC1 and AMPK, which phosphorylate ULK1 at distinct residues to either inhibit or enhance its function (19). The ULK family includes ULK1, ULK2, ULK3, ULK4, and STK368, with ULK1 and ULK2 showing the highest similarity (20). However, ULK1 is the dominant initiator of autophagy, as seen in \u003cem\u003eUlk1\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e \u003cem\u003eUlk2\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e double-knockout (DKO) mice, which exhibit severe neonatal mortality and autophagy defects similar to other core \u003cem\u003eAtg\u003c/em\u003e gene deletions (21-24). Although the cellular functions of ULK1 have been studied in several diseases, direct evidence of ULK1 role on tumor progression of PDAC, remains largely unexplored.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven the significant dependency of KRas-driven tumors including PDAC on autophagy (4, 7, 8, 25), pharmacological or genetic inhibition of KRas-downstream RAF, MEK, and ERK pathways has been shown to enhance autophagy, which supports the rationale for combining MAP kinase inhibitors with a derivative of chloroquine (CQ), a lysosomotropic agent (26-28). However, the therapeutic potential of CQ derivatives limited by the requirement for high inhibitory doses and poor selectivity, highlighting the need for more potent and selective autophagy modulators.\u003c/p\u003e\n\u003cp\u003eTargeting ULK1, an autophagy-initiating kinase, has emerged as an alternative strategy for autophagy inhibition. A series of ULK1 inhibitors have demonstrated antitumor effects \u003cem\u003ein vitro\u003c/em\u003e and in xenograft models (29, 30), and selective ULK1 inhibitors have shown synergistic tumor regression when combined with KRas or downstream effector inhibitors (31). However, despite the increasing interest in ULK1-targeted therapies, the exact role of ULK1 in cancer progression remains poorly understood.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we investigate how ULK1 function influences PDAC progression by employing syngeneic orthotopic and spontaneous cancer GEM models. Our findings reveal that tumor-intrinsic ULK1 deletion suppresses PDAC progression by impairing autophagy-mediated tumor adaptation and by reprogramming the tumor microenvironment through altered infiltration of distinct immune cell subtypes, thereby providing tumor-suppressive immune states. These results provide novel insights into ULK1 as a promising therapeutic target in pancreatic cancer.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMIA PaCa-2 and HEK293T cells were kindly provided by Drs. Yongdoo Choi and Jong Heon Kim (National Cancer Center Korea; NCC Korea), which are originally purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). KPC cell lines generous gift from Jong Heon Kim (NCC Korea), which are originally purchased from the Ximbio (London, UK), and were derived from PDAC tumors arising in KPC mice on a C57BL/6 background. All cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS; HyClone, Logan, UT), 100 U/mL penicillin, and 100 \u0026mu;g/mL of streptomycin (Gibco, Grand Island, NY), and maintained at 37 ℃ in a humidified incubator with 5 % CO2. For amino acid starvation media (-AA), Earle\u0026rsquo;s Balanced Salt Solution (EBSS, HyClone) or Hank\u0026rsquo;s balanced saline solution (HBSS) was supplemented with 10 % dialyzed FBS, glucose, vitamins, HEPES, and minerals at the same concentrations as in DMEM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of stable cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCRISPR-Cas9-mediated knockout of Ulk1 was performed lentiCRISPR v2 vector (a gift from Feng Zhang ;Addgene plasmid # 52961; RRID: Addgene_52961) (32).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003esgRNA oligo\u0026nbsp;targeting\u0026nbsp;mouse \u003cem\u003eU\u003c/em\u003e\u003cem\u003elk1\u003c/em\u003e CRISPR-Cas9 guide RNA (U0448BI200-1) or non-target sequence (sgControl) were synthesized (Gene Script, Piscataway, NJ). The guide RNA sequence were selected using the CRISPICK online tool (Broad institute, https://portals.broadinstitute.org/gppx/crispick/public) and sgRNA sequences are listed in Supplementary Table S2. Viral transduction processes were followed by standard protocols detailed in supplementary materials and methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHoechst 33342 (H3570) was purchased from Thermo Fisher Scientific\u0026nbsp;(Waltham, MA, USA). Rapamycin (R0395) was\u0026nbsp;purchased from Sigma-Aldrich\u0026nbsp;(St. Louis, MO, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eViability\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell proliferation was\u0026nbsp;monitored\u0026nbsp;using the image-based cell proliferation analyzer IncuCyte\u003csup\u003eTM\u003c/sup\u003e (Essen Instruments, Ann Arbor, MI, USA). Cells were\u0026nbsp;seeded\u0026nbsp;in complete DMEM media and imaged throughout the indicated time period. IncuCyte\u003csup\u003eTM\u003c/sup\u003e automated cell proliferation detector was used to measure cell confluence over time. Cell viability was determined by Annexin V and PI staining following standard protocols at the indicated time periods (556547;\u0026nbsp;BD Biosciences, San Jose, CA, USA), and analyzed\u0026nbsp;using the FACS Verse analyzer (BD Biosciences).\u0026nbsp;\u0026nbsp;Viability was assessed based on double-negative populations, and dead cells were defined by Annexin V⁺ and/or PI⁺ staining.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony formation\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSpheroid cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKPC cells\u0026nbsp;(5 x 10\u003csup\u003e2\u0026nbsp;\u003c/sup\u003ecells / well)\u0026nbsp;were\u0026nbsp;seeded in 12-well plates\u0026nbsp;in\u0026nbsp;complete\u0026nbsp;media (10 % FBS in DMEM). After 7 days, cells were fixed for 10 mins in 10 % formalin (HT501128-4L;\u0026nbsp;Sigma-Aldrich) and stained with 0.25 % crystal violet (C6158;\u0026nbsp;Sigma-Aldrich).\u0026nbsp;For\u0026nbsp;3D spheroids\u0026nbsp;culture,\u0026nbsp;KPC cells\u0026nbsp;(1 x 10\u003csup\u003e3\u0026nbsp;\u003c/sup\u003ecells /well)\u0026nbsp;were\u0026nbsp;seeded\u0026nbsp;in 96-well round-bottom Ultra Low Attachment (ULA) plates (7007;\u0026nbsp;Corning Inc., Corning, NY)\u0026nbsp;mixed with 1 % Matrigel (354234;\u0026nbsp;Corning Inc.)\u0026nbsp;and complete media.\u0026nbsp;Spheroid growth was\u0026nbsp;monitored\u0026nbsp;for 7 days and imaged using\u0026nbsp;HCS system Operetta CLS (PerkinElmer, Waltham, MA).\u0026nbsp;Quantification was performed by using Harmony software (PerkinElmer).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003cstrong\u003envasion assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInvasion assay was performed to ascertain cell invasion using 0.8\u0026thinsp;\u0026mu;m Transwell apparatus (Corning Inc.) with\u0026nbsp;coated\u0026nbsp;Matrigel (354234;\u0026nbsp;Corning Inc.) on a 24-well plate. KPC cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) suspended in 100 \u0026mu;l serum-free medium were seeded into the upper chamber\u0026nbsp;and\u0026nbsp;complete DMEM\u0026nbsp;was\u0026nbsp;added to the lower chamber. After incubation at 37 \u0026deg;C for 24 h, invaded\u0026nbsp;cells were stained using Diff-Quik reagents (Sysmex Corporation, Kobe, Japan)\u0026nbsp;and counted under an inverted microscope at a 4\u0026nbsp;\u0026times; and 20\u0026nbsp;\u0026times; magnifications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLSL\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e-\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eKras\u003csup\u003eG12D/+\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e; \u003cem\u003eLSL\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e-\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eTrp53 \u003csup\u003eR172H/+\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e; \u003cem\u003ePdx1-Cre\u003c/em\u003e;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eUlk1\u003csup\u003efl\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u003csup\u003e/\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u003csup\u003efl\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eGenetically Engineered Mouse Model\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eKras\u003csup\u003eG12D/+\u003c/sup\u003e\u003c/em\u003e (B6.129S4-\u003cem\u003eKras\u003csup\u003etm4Tyj\u003c/sup\u003e\u003c/em\u003e, strain #01XJ6) was received from the NCI mouse repository (NCI, Bethesda, MD, USA).\u0026nbsp;\u003cem\u003eTrp53\u003csup\u003eR172H/+\u003c/sup\u003e\u003c/em\u003e (129S-\u003cem\u003eTrp53\u003csup\u003etm2Tyj\u003c/sup\u003e\u003c/em\u003e/J, strain #008652) and \u003cem\u003ePdx1\u003c/em\u003e-Cre (B6.FVB-Tg (\u003cem\u003ePdx1\u003c/em\u003e-Cre)6Tuv/J, strain #014647) mouse were purchased from the Jackson Laboratory\u0026nbsp;(Bar Harbor, ME, USA).\u0026nbsp;These mice were backcrossed more than six times with C57BL/6 and were subsequently used for generating KPC (\u003cem\u003eKras\u003csup\u003eG12D/+\u003c/sup\u003e\u003c/em\u003e; \u003cem\u003eTrp53\u003csup\u003eR172H/+\u003c/sup\u003e\u003c/em\u003e; \u003cem\u003ePdx1\u003c/em\u003e-Cre) mouse. The KPC model of PDAC was first described in 2005 and incorporates, through Cre-lox technology, the conditional activation of mutant endogenous alleles of the \u003cem\u003eKras\u003c/em\u003e and \u003cem\u003eTrp53\u003c/em\u003e gene (33).\u0026nbsp;\u003cem\u003eUlk1\u003c/em\u003e\u003csup\u003efl\u003c/sup\u003e\u003csup\u003e/\u003c/sup\u003e\u003csup\u003efl\u003c/sup\u003e (B6.129-\u003cem\u003eUlk1\u003csup\u003etm1Thsn\u003c/sup\u003e\u003c/em\u003e/J, stock #017976) mice were purchased from The Jackson Laboratory (Sacramento, CA), which were generated and donated by Craig B. Thompson (Memorial Sloan Kettering Cancer Center, NY, USA)\u0026nbsp;(24). The \u003cem\u003eUlk1\u003c/em\u003e\u003cem\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e and KPC mice were crossed, and after several rounds of breeding, \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003c/em\u003e\u003cem\u003e\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice were successfully obtained.\u0026nbsp;All animal procedures were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the NCC. NCC is an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International)-accredited facility that abides by the guidelines of the Institute of Laboratory Animal Resources (ILAR) Guide and Usage Committee.\u0026nbsp;The methods applied in this study were performed in accordance with the approved guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOrthotopic\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003cstrong\u003eancreatic\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cstrong\u003eancer mouse model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix-week-old female C57BL/6 mice (Orient Bio Inc., Seongnam, Korea) were orthotopically injected with 2 \u0026times; 10⁵ KPC\u0026nbsp;sgSC or KPC\u0026nbsp;sg\u003cem\u003eUlk1\u003c/em\u003e cells suspended in 1:1 Matrigel (50 \u0026micro;L) into the pancreatic parenchyma.\u0026nbsp;After injection, the peritoneum and skin were closed with a 6-0 suture (639G;\u0026nbsp;Ethicon, Raritan, NJ).\u0026nbsp;At 3 weeks later of allograft, pancreas\u0026nbsp;was\u0026nbsp;isolated from the mice and\u0026nbsp;used for indicated experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological and Immunohistochemical Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePancreas tissues\u0026nbsp;from indicated allografted mice or pancreatic tissues from \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003e+/fl\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e GEM mice\u003cem\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003ewere isolated from 12\u0026nbsp;weeks to 17 weeks old mice, respectively. The tissues were fixed in\u0026nbsp;10%\u0026nbsp;Neutral Buffered Formalin (BN019,\u0026nbsp;Biosolution, Seoul, Korea) and paraffin-embedded.\u0026nbsp;Section (4\u0026thinsp;\u0026mu;m thick) of mouse pancreas were deparaffinized, rehydrated and incubated in boiling CC1(pH 9) or CC2(pH 6) for antigen retrieval by Benchmark TX (950-123 or 124; Ventana Medical Systems, Oro Valley, AZ) or Discovery XT (Ventana Medical Systems).\u003c/p\u003e\n\u003cp\u003eImmunohistochemical\u0026nbsp;(IHC)\u0026nbsp;staining\u0026nbsp;was performed\u0026nbsp;with the indicated primary antibodies\u0026nbsp;(Supplementary Table S1)\u0026nbsp;and\u0026nbsp;then\u0026nbsp;detected\u0026nbsp;with a DAB detection kit (Ventana Medical Systems) according to the manufacturer\u0026rsquo;s instructions, followed by counterstaining with hematoxylin (Ventana Medical Systems).\u0026nbsp;The stained\u0026nbsp;images were\u0026nbsp;acquired using the\u0026nbsp;Vectra Polaris (Perkin Elmer/AKOYA Biosciences)\u0026nbsp;and\u0026nbsp;quantified\u0026nbsp;using inForm\u003csup\u003eⓡ\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eTissue Analysis Software (AKOYA Biosciences, Marlborough, MA). H-Score = (1 X % of 1+ staining spot) + (2 X % of 2+ staining spot) + (3 X % of 3+ staining spot). Statistical analysis was calculated using Graphpad Prism 8.0.1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMultiplex\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003cstrong\u003emmunohistochemistry (multi-IHC) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTMA sections from PDAC patients (PA241e, TissueArray.Com LLC, Derwood, MD, USA) and the FFPE pancreas tissue from KPC \u003cem\u003eUlk1\u003csup\u003e+/fl\u003c/sup\u003e\u003c/em\u003e and KPC \u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice were subjected to multiplex immunohistochemistry (multi-IHC) with indicated antibodies (Supplementary Table S1) and automated staining Leica Bond RX (Leica Biosystems, Vista, CA) using Opal 7 kit (#NEL871001KT, #ARR1001KT; Akoya Biosciences).\u0026nbsp;Tyramide signal amplification (TSA) fluorophores were used to combine with each antibody separately according to manufacturer\u0026rsquo;s instructions.\u0026nbsp;Imaging and quantification\u0026nbsp;were\u0026nbsp;performed by Vectra Polaris Automated Quantitative Pathology Imaging System (Perkin Elmer Inc.) and inForm image analysis software (Akoya Biosciences). \u0026nbsp;Data were compiled and\u0026nbsp;analyzed after it merged and consolidated each case by R studio\u0026nbsp;(version\u0026nbsp;2021.09.2.0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cstrong\u003eytometry for\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Tumor\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Immunophenotyping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the immunophenotyping of tumor,\u0026nbsp;tumor\u0026nbsp;tissues were dissociated in a fully automated way by using the gentleMACS\u003csup\u003eTM\u003c/sup\u003e Octo Dissociator with Heaters (130-096-427;\u0026nbsp;Miltenyi\u0026nbsp;Biotec,\u0026nbsp;Bergisch Gladbach,\u0026nbsp;Germany) with collagenase P (C7657,\u0026nbsp;Merk) and the Tumor Dissociation Kit (130-096-730;\u0026nbsp;Miltenyi\u0026nbsp;Biotec), which are optimized for epitope preservation. Digested tissues were filtered through a 70 \u0026mu;m nylon cell strainer (352350; BD\u0026nbsp;Falcon,\u0026nbsp;Franklin Lakes, NJ). Cells were blocked with anti-mouse CD16/CD32 antibody (Mouse BD Fc Block\u003csup\u003eTM\u003c/sup\u003e) (BD553142, clone 2.4G2;\u0026nbsp;BD Biosciences) and\u0026nbsp;stained with indicated antibodies\u0026nbsp;for 20 min at 4 \u0026deg;C. Intracellular staining was performed using Fixation/Permeabilization Kit (BD554714;\u0026nbsp;BD Bioscience). Samples were analyzed on a LSRFortessa\u0026trade; Cell Analyzer (BD Biosciences).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokine array\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCytokine profiles\u0026nbsp;were assessed\u0026nbsp;using\u0026nbsp;the\u0026nbsp;Proteome Profiler Mouse Cytokine Array Kit (ARY006; R\u0026amp;D systems, Minneapolis, MN, USA) following the manufacturer\u0026rsquo;s instruction. Detailly, mouse pancreatic cancer tissues were homogenized by\u0026nbsp;Tissue Lyser II (QIAGEN, Hilden, Germany) in\u0026nbsp;PBS containing protease inhibitor cocktail (Roche, Basel, Switzerland).\u0026nbsp;and then lysed with 1%\u0026nbsp;Triton X-100. Cellular debris was removed by centrifugation at 10,000xg for 5 min at\u0026nbsp;4\u0026nbsp;℃\u0026nbsp;and\u0026nbsp;protein was\u0026nbsp;quantified using BCA Protein Assay (23227;\u0026nbsp;Thermo Fisher Scientific\u0026nbsp;Inc.). Conditioned media were collected from cells cultured under identical conditions for 24 hours. Media were then normalized to the total cellular protein content, as determined from corresponding cell lysates. Membranes incubated with conditioned media were developed and visualized using the FUSION SOLO S imaging system (Vilber, Coll\u0026eacute;gien, France) with exposure times ranging from 2 to 6 minutes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmune\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cstrong\u003eell\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eV\u003c/strong\u003e\u003cstrong\u003eiability analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePancreas, spleen and blood from the abdominal aorta were collected and dissociated as indicated above.\u0026nbsp;PMN-MDSC/neutrophil (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e),\u0026nbsp;CD8 T cell (CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e)\u0026nbsp;and DCs (CD45\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e) were sorted\u0026nbsp;using Melody (BD Biosciences),\u0026nbsp;and then cultured in the presence of 50% conditioned media from\u0026nbsp;KPC sgSC or KPC_sg\u003cem\u003eUlk1\u003c/em\u003e cells\u0026nbsp;on\u0026nbsp;a 384- or 96-well black well plate (6057308 and 6055302; PerkinElmer).\u0026nbsp;After incubation\u0026nbsp;with\u0026nbsp;indicated time,\u0026nbsp;live cell numbers were assessed using\u0026nbsp;Calcein-AM (C1430; Invitrogen, Carlsbad, CA)\u0026nbsp;staining. Imaging and quantification were performed using the Operetta CLS system and Harmony software\u0026nbsp;(PerkinElmer).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatic\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Cancer Genome Atlas\u0026nbsp;(TCGA)-pancreatic adenocarcinoma (PAAD)\u0026nbsp;patients were stratified\u0026nbsp;with the autophagy score\u0026nbsp;using the surv_cutpoint function from the survminer package (version 0.4.9). Kaplan-Meier overall survival plot\u0026nbsp;and the p-value between patient groups was calculated using the Log-Rank test implemented in the survival package (version 3.5-7). Cox Hazard ratio and confidence interval was calculated using coxph function of the survival package.\u003c/p\u003e\n\u003cp\u003eThe autophagy score was calculated using a modified version of ssGSEA2.0, which is an adaptation of the GSEA algorithm for single-cell analysis (34, 35). The list of genes used for the autophagy score calculation can be found in the supplementary information. Default parameters for ssGSEA were used to calculate scores for TCGA-PAAD patients and for cells in single-cell data from pancreatic cancer patients (CRA001160) (35, 36).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor gene set enrichment analysis, TCGA database were accessed in the Cancer Integrative Platform (cBioportal; http://www.cBioportal.mskcc.org/). For the survival analysis, the latest TCGA iteration of transcriptome and clinical data was downloaded directly using the application programming interface provided by TCGAbiolinks (version 2.30.4) in R (4.3.1). All other datasets supporting the current study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmuno-blot quantifications were performed using\u0026nbsp;Image J software version 1.50i (NIH, Bethesda, MD). Statistical significance was calculated using Student\u0026rsquo;s t test in Graph Pad Prism 8. A value of p \u0026lt; 0.05 was considered statistically significant (* p \u0026lt; 0.05; ** p \u0026lt; 0.01; *** p \u0026lt; 0.001).\u0026nbsp;Data are expressed as standard error of the mean (SEM) which are from at least\u0026nbsp;three\u0026nbsp;independent experiments.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eULK1 Activity is Elevated in Pancreatic Cancer Independent of mRNA Expression\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To assess the clinical relevance of autophagy proteins in pancreatic cancer, we analyzed the expression of 25 core autophagy proteins in pancreatic adenocarcinoma (PAAD)\u0026nbsp;from The Cancer Genome Atlas (TCGA).\u0026nbsp;These autophagy gene sets were significantly upregulated in tumor tissues compared to adjacent normal tissues\u0026nbsp;(P \u0026lt; 0.05;\u0026nbsp;Supplementary Fig. S1A\u0026ndash;C), and\u0026nbsp;high\u0026nbsp;expression levels of autophagy gene sets correlated\u0026nbsp;with\u0026nbsp;poor\u0026nbsp;5-year overall survival\u0026nbsp;(Supplementary Fig. S1D),\u0026nbsp;supporting the tumor-promoting role of autophagy in PDAC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInterestingly, the mRNA levels of \u003cem\u003eULK1\u003c/em\u003e was not significantly different between tumor and normal tissues (Supplementary Fig. S1E).\u0026nbsp;However, multiplex immunohistochemistry (multi-IHC) revealed elevated ULK1 protein levels and activity, marked by phosphorylated ATG14 (pATG14), particularly in high-grade (Grade 3) pancreatic tumors compared to normal tissues (Fig. 1A\u0026ndash;D, Supplementary Fig. S2A, B).\u003c/p\u003e\n\u003cp\u003eSimilarly, increases in ULK1 and pATG14 levels were observed in human pancreatic cancer cell lines compared to normal human pancreatic epithelial (HPNE) and fibroblast (IMR90) cells (Fig. 1E). Moreover, elevated expression of ULK1 and pATG14 was detected in transformed pancreatic intraepithelial neoplasia (PanIN) in\u0026nbsp;\u003cem\u003eKras\u003csup\u003e+/LSL-G12D\u003c/sup\u003e;\u003c/em\u003e \u003cem\u003ePdx1-Cre\u003c/em\u003e (KC)\u0026nbsp;mice and\u0026nbsp;in advanced\u0026nbsp;PDAC lesions\u0026nbsp;from\u0026nbsp;\u003cem\u003eKras\u003csup\u003e+/LSL-G12D\u003c/sup\u003e;\u003c/em\u003e \u003cem\u003eTrp53\u003csup\u003eR172H/+\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e;\u003c/em\u003e \u003cem\u003ePdx1-Cre\u003c/em\u003e (KPC) mice (Fig. 1F).\u0026nbsp;These\u0026nbsp;results\u0026nbsp;suggest that\u0026nbsp;ULK1\u0026nbsp;activity\u0026nbsp;rather than its mRNA levels\u0026nbsp;is significantly elevated in malignant pancreatic cancers, supporting its potential role in tumor progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eULK1 Depletion Suppresses Pancreatic Cancer Cell Proliferation, Invasion, and Autophagy\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo\u0026nbsp;investigate\u0026nbsp;the functional role of ULK1 in pancreatic cancer progression, we generated \u003cem\u003eUlk1\u003c/em\u003e knockout (KO) KPC cells using CRISPR-Cas9, which was confirmed by genomic sequencing and qRT-PCR (Supplementary Fig. S3A, B). \u003cem\u003eUlk1\u003c/em\u003e KO cells exhibited significantly impaired cell growth compared to control cells, as measured by IncuCyte live-cell imaging (Fig. 2A) and MTT assay (Fig. 2B). ULK1 loss also reduced colony formation (Fig. 2C) and markedly impaired 3D spheroid growth (Fig. 2D, E). Additionally, Transwell invasion assay\u0026nbsp;showed\u0026nbsp;a\u0026nbsp;substantial\u0026nbsp;decrease in invasion of \u003cem\u003eUlk1\u003c/em\u003e KO cells compared to sgSC controls (Fig. 2F, G), indicating a significant reduction in both proliferative and invasive potential.\u003c/p\u003e\n\u003cp\u003eTo determine whether these phenotypes were linked to autophagy, we assessed autophagic flux by analyzing LC3 processing and localization. ULK1-deficient cells accumulated LC3-I with reduced conversion to LC3-II, particularly under amino acid-deprived conditions (Fig. 2H), and confirmed by a lower LC3-II/I ratio in Bafilomycin A1-treated cells (Supplementary Fig. S3C, D). Rapamycin-induced GFP-LC3 puncta formation was also significantly impaired in ULK1 knockdown cells (Fig. 2I, J) and, further confirming autophagy suppression and defect of viability (Supplementary Fig. S3E).\u0026nbsp;These findings indicate that ULK1 is required for nutrient-responsive autophagy, and that its loss impairs both growth and invasion of pancreatic cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eULK1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;Loss\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;Delays Tumor Progression in\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eOrthotopic\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ePancreatic Tumor\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eModel\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo extend our \u003cem\u003ein vitro\u003c/em\u003e findings, we investigated the\u0026nbsp;\u003cem\u003ein vivo\u003c/em\u003e role of ULK1 in tumor progression using syngeneic mouse model. An orthotopic PDAC mouse model\u0026nbsp;was\u0026nbsp;established\u0026nbsp;by injecting \u003cem\u003eUlk1\u003c/em\u003e KO (sg\u003cem\u003eUlk1\u003c/em\u003e) or control (sgSC)\u0026nbsp;KPC\u0026nbsp;cells into the pancreas of C57BL/6J mice. Histological analysis 3\u0026ndash;5 weeks post-injection revealed reduced Ki67 staining in ULK1-deficient tumors compared to controls, consistent with decreased Ulk1 expression\u0026nbsp;(Fig.\u0026nbsp;2K, L).\u0026nbsp;Moreover, mice bearing \u003cem\u003eUlk1\u003c/em\u003e KO tumor showed significantly\u0026nbsp;extended survival compared\u0026nbsp;to\u0026nbsp;control\u0026nbsp;KPC\u0026nbsp;tumor-injecting mice\u0026nbsp;(Fig.\u0026nbsp;2M). These findings support a\u0026nbsp;critical role of Ulk1 in promoting cell proliferation and tumor progression in allograft \u003cem\u003ein vivo\u003c/em\u003e model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eUlk1 Deficiency Suppresses Pancreatic Tumor Development in a Spontaneous Mouse Model\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the role of ULK1 in pancreatic cancer progression, we utilized the \u003cem\u003eLSL\u003c/em\u003e\u003cem\u003e-\u003c/em\u003e\u003cem\u003eKras\u003csup\u003eG12D/+\u003c/sup\u003e;\u0026nbsp;LSL\u003c/em\u003e\u003cem\u003e-\u003c/em\u003e\u003cem\u003eTrp53\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003e\u003csup\u003eR172H/+\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e;\u0026nbsp;\u003c/em\u003e\u003cem\u003ePdx1-Cre\u003c/em\u003e (KPC) mouse model (33), which spontaneously develops pancreatic tumors that mimic the pathology of human PDAC. These mice were crossbred with previously established \u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (24) to generate \u003cem\u003eKPC;Ulk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice, enabling pancreas-specific \u003cem\u003eUlk1\u003c/em\u003e deletion via Cre recombinase (Fig. 3A). In this genetically engineered mouse (GEM) model, the pancreas-specific expression of Cre recombinase, driven by \u003cem\u003ePdx1\u003c/em\u003e-Cre, induces a constitutively active \u003cem\u003eKras\u003c/em\u003e mutation (G12D) and a dominant negative \u003cem\u003eTrp53\u003c/em\u003e mutation (R172H) (Fig. 3A; Supplementary Fig. S4A). Immunoblotting confirmed efficient Cre-mediated \u003cem\u003eUlk1\u003c/em\u003e deletion in pancreatic tissues (Fig. 3B). Histological analysis showed only significant abnormalities in pancreas rather than other tissues, though occasional lung pathology was observed in positive control (\u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e) mice (Fig. 3C) suggesting \u003cem\u003eUlk1\u003c/em\u003e deletion specifically impacts pancreatic tumor development. However, pancreas weights were significantly reduced in \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice compared to that of KPC control mice (\u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e) (Fig. 3D). Moreover, 18F-FDG-PET/CT imaging revealed a reduced tumor burden and lower SUVmax values in \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003emice, compared to \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003econtrol mice (Fig. 3E; Supplementary Fig. S4B). Notably, increased FDG uptake in the lungs, indicative of distant metastasis, was observed in \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003econtrol mice but diminished in \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003emice.\u0026nbsp;These findings suggest that \u003cem\u003eUlk1\u003c/em\u003e deletion primarily impacts pancreatic tumor development in the KPC model, with occasional lung metastasis in late stages of cancer.\u003c/p\u003e\n\u003cp\u003eNext, further histopathological results from pancreas showed that all KPC control mice (\u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eor \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003e+/+\u003c/sup\u003e\u003c/em\u003e) developed moderate-to-malignant PDAC within 18 weeks, whereas only 12 % (1/8) \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (pancreas-specific \u003cem\u003eUlk1\u003c/em\u003e KO KPC) developed low -grade PanIN lesions, with the majority displaying normal pancreatic architecture with healthy acini cells (Fig. 4A, B). Immunohistochemistry (IHC) staining further revealed significantly lower levels for cytokeratin 19 (CK19) and Ki67 in \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e pancreatic tissues compared to those from KPC control (\u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e) mice, consistent with suppressed epithelial transformation and proliferative capacity (Fig. 4C, D). These findings aligned with reduced expression\u0026nbsp;of Ulk1 and pAtg14 in the pancreatic ductal regions of \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003emice, which was more pronounced in the transformed regions of control KPC mice (Fig. 4C, D). In agreement with impaired autophagic flux, LC3B, an autophagy marker, was significantly decreased in the pancreas of \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e(Supplementary Fig. S4C, D).\u003c/p\u003e\n\u003cp\u003eMultiplex-IHC further confirmed that Ulk1 and pAtg14 levels were significantly enriched in Ck19\u003csup\u003e\u0026nbsp;+\u003c/sup\u003e epithelial cells within tumor from \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e mice, while these signals were nearly absent in the tissue from \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (Fig. 4E, F). Importantly, \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice exhibited a significantly prolonged median overall survival\u0026mdash; approximately 10 weeks longer than that of \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e controls (Fig. 4G).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSpatial analysis supported a strong association between CK19⁺ adenocarcinoma cells in and pAtg14⁺ autophagy-active cells in \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e tumors, which was largely diminished in Ulk1 deficient mice (Fig. 4H, I). Collectively, these data demonstrate that Ulk1 is critical for PDAC progression in a physiologically relevant spontaneous PDAC model and support its function as a key effector of both primary tumor growth and metastatic potential.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eUlk1 Knockout Alters Tumor Immune Microenvironment and Enhances Antitumor Immunity\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the role of autophagy in modulating tumor immunity (37, 38), we next examined whether \u003cem\u003eUlk1\u003c/em\u003e deletion alters immune cell composition within the tumor microenvironment (TME). Immunohistochemical analysis revealed that several TME markers increased tumor-associated fibroblasts (\u0026alpha;SMA) and immunosuppressive M2 macrophages (CD204) in \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e control mice tumor, particularly in CK19\u003csup\u003e+\u003c/sup\u003e adenocarcinoma lesions, whereas these markers were significantly reduced in the pancreas of \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emice (Fig. 5A, B). Immunosuppressive immune cells neutrophils (Ly6G) were also stained stronger in \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e control mice tumor rather than reduced in the pancreas of \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emice. In contrast, markers for tumor-infiltrating lymphocytes, such as cytotoxic T cells (CD8ɑ) and natural killer (NK) cells (NCR1), were more abundant in pancreas of \u003cem\u003eUlk1\u003c/em\u003e KO mice (\u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e) than in control KPC mice (\u003cem\u003eUlk1\u003c/em\u003e wild-type (WT) or heterozygous KPC (\u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e), suggesting an enhanced antitumor immune response (Fig. 5A, B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMultiplex IHC further confirmed that, CD204\u003csup\u003e+\u003c/sup\u003e M2 macrophages were more abundant in control tumor (\u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e) mice, while CD8\u003csup\u003e+\u003c/sup\u003e T cells were more enriched in\u003cem\u003e\u0026nbsp;\u003c/em\u003epancreas from \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice\u003cem\u003e\u0026nbsp;\u003c/em\u003ethan in\u0026nbsp;control KPC\u0026nbsp;mice, although statistical significance were not shown (Supplementary Fig. 5A\u0026ndash;C).\u003c/p\u003e\n\u003cp\u003eTaken together, these findings indicate that \u003cem\u003eUlk1\u003c/em\u003e deletion remodels the pancreatic TME by reducing immunosuppressive immune cells and increasing cytotoxic lymphocyte infiltration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eULK1 Deletion Reshapes Immune Cell Composition in Pancreatic Tumors\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the molecular mechanisms underlying tumor regression following \u003cem\u003eUlk1\u003c/em\u003e depletion, we performed RNA-sequencing analysis of \u003cem\u003eUlk1\u003c/em\u003e WT and \u003cem\u003eUlk1\u003c/em\u003e KO KPC cells. Differential expression analysis identified 4,097 genes with altered expression including 1071 upregulated and 1538 downregulated in \u003cem\u003eUlk1\u003c/em\u003e KO cells. KEGG pathway enrichment analysis of upregulated gene sets in \u003cem\u003eUlk1\u003c/em\u003e KO cells revealed significant enrichment \u0026ldquo;MAPK signaling\u0026rdquo;, \u0026ldquo;PI3K-Akt signaling\u0026rdquo; and \u0026ldquo;Cytokine-cytokine receptor interaction\u0026rdquo; pathways (Supplementary Table S3). Proteomic profiling of \u003cem\u003eKRas\u003csup\u003eG12D\u003c/sup\u003e\u003c/em\u003e-expressing HPNE cells treated with chloroquine (CQ) revealed a similar upregulation of immune-related pathways, with \u0026ldquo;Cytokine\u0026ndash;cytokine receptor interaction\u0026rdquo;, \u0026ldquo;JAK\u0026ndash;STAT signaling\u0026rdquo;, \u0026ldquo;TGF-\u0026beta; signaling\u0026rdquo;, and \u0026ldquo;Mitophagy\u0026rdquo; among the most enriched pathways (Supplementary Table S4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eComplementing these results, gene set enrichment analysis (GSEA) of TCGA pancreatic cancer datasets (34) revealed that lower \u003cem\u003eULK1\u003c/em\u003e expression correlated with elevated immune and mitochondrial gene signatures. Gene ontology (GO) analysis of gene clusters negatively co-expressed with \u003cem\u003eULK1\u003c/em\u003e revealed strong enrichment for terms related to \u0026ldquo;Antigen processing and presentation\u0026rdquo;, \u0026quot;Immune system processes\u0026quot;, and \u0026ldquo;Mitochondrial metabolism\u0026rdquo;, suggesting a link between \u003cem\u003eULK1\u003c/em\u003e suppression and activation of immune and mitochondria metabolic pathways (Supplementary Table S5).\u003c/p\u003e\n\u003cp\u003eTo further assess whether tumor-intrinsic \u003cem\u003eUlk1\u003c/em\u003e deletion affects immune cell composition \u003cem\u003ein vivo\u003c/em\u003e, we employed syngeneic orthotopic models by injecting KPC sgSC (\u003cem\u003eUlk1\u003c/em\u003e WT) and KPC sg\u003cem\u003eUlk1\u003c/em\u003e (\u003cem\u003eUlk1\u003c/em\u003e KO) cells into the pancreas of immunocompetent C57BL/6J mice. After 3 weeks, the mice were sacrificed and subjected to immunophenotyping by FACS analysis using the gating strategy shown in Supplementary Fig. S6.\u003c/p\u003e\n\u003cp\u003eAlthough the overall proportions of tumor-infiltrating CD45\u003csup\u003e+\u003c/sup\u003e immune cells and CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e myeloid cells remained unchanged between the two groups, \u003cem\u003eUlk1\u003c/em\u003e KO-derived tumor showed a marked reduction in the proportion of neutrophils (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs; CD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e\u0026minus;\u003c/sup\u003e) compared with tumors derived from \u003cem\u003eUlk1\u003c/em\u003e WT KPC cells, alongside a significant increase in MHC class II\u003csup\u003e+\u003c/sup\u003e antigen-presenting cells (APCs) (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e MHC-II\u003csup\u003e+\u003c/sup\u003e) in tumor derived from \u003cem\u003eUlk1\u003c/em\u003e KO KPC cells (Fig. 6A, B).\u003c/p\u003e\n\u003cp\u003eIn the lymphoid compartment, \u003cem\u003eUlk1\u003c/em\u003e KO cell-derived tumors exhibited higher proportion of CD8\u003csup\u003e+\u003c/sup\u003e T cells over CD4\u003csup\u003e+\u003c/sup\u003e T cell ratio than in \u003cem\u003eUlk1\u003c/em\u003e WT tumors, though total CD3\u003csup\u003e+\u003c/sup\u003e or CD4\u003csup\u003e+\u003c/sup\u003e T cell proportions were unchanged (Supplementary Fig. S6B, S7A). This increase in cytotoxic T cells may be linked to enhanced recruitment of MHC class II\u003csup\u003e+\u003c/sup\u003e APCs in \u003cem\u003eUlk1\u003c/em\u003e KO tumors.\u003c/p\u003e\n\u003cp\u003eTo validate these findings in a spontaneous PDAC model, we performed similar immunophenotyping in KPC control and \u003cem\u003eKPC;Ulk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (Fig. 6C, D). Similar to the orthotopic model, we observed increased proportions of MHC II\u003csup\u003e+\u003c/sup\u003e APC cells, and significantly reduced neutrophils and PMN- in \u003cem\u003eKPC;Ulk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice. In lymphoid populations, CD8\u003csup\u003e+\u003c/sup\u003e T cell proportions were considerably elevated in \u003cem\u003eKPC;Ulk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice, whereas the CD4\u003csup\u003e+\u003c/sup\u003e T cell proportion remained comparable to that in \u003cem\u003eKPC;Ulk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003econtrol mice, consistent with the results of the syngeneic orthotopic model (Supplementary Fig. S7B).\u003c/p\u003e\n\u003cp\u003eCollectively, these data from two independent \u003cem\u003ein vivo\u003c/em\u003e models indicate that tissue-specific \u003cem\u003eUlk1\u003c/em\u003e depletion enhances antitumor immune responses by markedly reducing immuno-suppressive PMN-MDSCs and neutrophils while promoting APCs and CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration, thus contributing to tumor regression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eUlk1 regulates Cytokines and Chemokine Secretion to Support a Pro-Tumorigenic Immune Microenvironment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further dissect how ULK1 shapes the immune landscape in pancreatic tumors, we profiled cytokine and chemokine expression in pancreatic tissues from KPC control (\u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/+\u003c/sup\u003e\u003c/em\u003e) and KPC \u003cem\u003eUlk1\u003c/em\u003e KO (\u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e) mice. Several pro-tumorigenic cytokines and chemokines including ICAM-1/CD54, TIMP1, IL-1RN (IL-1ra), and IL-16 (34, 39-41) were upregulated in KPC control pancreatic tissues compared with those in \u003cem\u003eKPC\u003c/em\u003e;\u003cem\u003eUlk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (Supplementary Fig. 8A, B). While Ccl2 was modestly downregulated in \u003cem\u003eKPC;Ulk1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e tissues, Cxcl12 levels remained unchanged.\u003c/p\u003e\n\u003cp\u003eCytokine and chemokine profiling of conditioned media (CM) from \u003cem\u003eUlk1\u003c/em\u003e WT and \u003cem\u003eUlk1\u003c/em\u003e KO KPC cells revealed distinct secretory profiles, reinforcing the tumor-promoting immune landscapes (Fig. 7A, B). While Icam-1/CD54 was remained undetectable, and Timp1 levels were comparable in CM from KPC sgSC and KPC sg\u003cem\u003eUlk1\u003c/em\u003e cells, suggesting these chemokines originate from fibroblasts rather than from tumor epithelial and immune cells. Pro-tumorigenic chemokines Cxcl2 and Ccl2, which promoting recruitment of PMN-MDSCs and M2 macrophages (42), were substantially reduced in \u003cem\u003eUlk1\u003c/em\u003e KO cells, consistent with previous immunophenotyping data from pancreatic tissues from spontaneous cancer model (Fig. 6). Additionally, the secreted levels of G-CSF and Ccl2 were notably reduced, whereas GM-CSF levels were markedly elevated in in CM from \u003cem\u003eUlk1\u003c/em\u003e KO cells, indicating that ULK1 determines to regulate tumor-intrinsic G-CSF, Ccl2 and GM-CSF secretion oppositely (Fig. 7A, B). These findings were confirmed by qPCR, which reduced expression of \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eIL-1rn\u003c/em\u003e, \u003cem\u003eCxcl2\u003c/em\u003e, and \u003cem\u003eCsf3\u003c/em\u003e (G-CSF) in \u003cem\u003eUlk1\u003c/em\u003e KO cells but increased \u003cem\u003eCsf2\u003c/em\u003e (GM-CSF) expression (Fig. 7C). ELISA results also exhibited the concentration of Ccl2, G-CSF and GM-CSF secreted from both genotyped cells, which were consistent with their gene expression levels (Fig. 7D).\u003c/p\u003e\n\u003cp\u003eTogether, these data demonstrate that Ulk1 depletion suppresses secretion of key cytokines and chemokines that support the recruitment of tumor-promoting immune cell subsets. However, Ulk1 loss oppositely increased a crucial cytokine such as GM-CSF that promotes the tumor-suppressive immune cell populations, through tumor-intrinsic signaling, ultimately leading to a less immune-suppressive TME and further reduced tumor progression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eULK1-Dependent Cytokine Signaling Influences Immune Cell Survival\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether tumor-derived factors directly influence tumor-infiltrated immune cell survival, we isolated freshly immune cells from either pancreas tumor tissues or blood/spleen of tumor-bearing KPC mice at 15 weeks old and sorted neutrophils including PMN-MDSC (CD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e), cytotoxic T cells (CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e) and dendritic cells (DCs; CD45\u003csup\u003e+\u003c/sup\u003e CD11c\u003csup\u003e+\u003c/sup\u003e), respectively. These sorted immune cells were cultured in CM from either \u003cem\u003eUlk1\u003c/em\u003e-WT or KO KPC cells for 24 h and then assessed their viability by Calcein-AM staining. Neutrophils including PMN-MDSCs from tumors showed significantly higher viability cultured in CM from KPC WT cells (Fig. 7E; Supplement Fig. S9A). In contrast, the viability of CD8\u003csup\u003e+\u003c/sup\u003e T cells and dendritic cells (DC) from tumor was significantly higher in \u003cem\u003eUlk1\u003c/em\u003e KO CM than in KPC WT (Fig. 7 E; Supplement Fig. S9A). Interestingly, these viability differences were not observed in immune cells isolated from blood or spleen of tumor-bearing KPC mice when cultured in the same CM, suggesting that Ulk1-dependent tumor -secreted factors specifically affect immune cells within the tumor context (Supplement Fig. S9B, C).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollectively, these results reveal that ULK1 orchestrates a tumor-intrinsic cytokine/chemokine program that shapes an immunosuppressive microenvironment by supporting the survival of neutrophils including PMN-MDSCs and suppressing DC and cytotoxic T cell function\u0026mdash;mechanisms which are disrupted upon ULK1 deletion.\u003c/p\u003e\n\u003cp\u003eFinally, to evaluate the translational relevance of these findings, we analyzed tissue microarrays (TMAs) from human PDAC samples. Grade 3 adenocarcinoma regions displayed higher CD163\u003csup\u003e+\u003c/sup\u003e M2 macrophage staining, while the infiltration of CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells and MHC II\u003csup\u003e+\u003c/sup\u003e APCs was lower in tumor lesions (Adeno) than in adjacent normal tissue (CTL) (Fig. 7F, G). This staining pattern was also observed in TMA with grade 1 and 2 (Supplementary Fig. S10A, B), consistent with high ULK1 activity observed in human pancreatic cancers (Fig. 1A and Supplementary Fig. S2A).\u003c/p\u003e\n\u003cp\u003eTaken together, these findings indicate that \u003cem\u003eUlk1\u003c/em\u003e deletion remodels the tumor immune microenvironment by reducing tumor-promoting myeloid cell such as M2 macrophages and neutrophils/PMN-MDSCs, and increasing cytotoxic T cells and APCs, leading to enhanced antitumor immunity and suppressed tumor growth.\u0026nbsp;\u003c/p\u003e\n"},{"header":"Discussion","content":"\u003cp\u003eAutophagy plays a context-dependent role in cancer, functioning as a tumor suppressor during early tumorigenesis but promoting tumor survival in established malignancies. In PDAC, elevated autophagy activity supports tumor progression by providing essential metabolic substrates for cancer cell survival. Although the importance of autophagy in PDAC has been demonstrated by the tumor-suppressive effects of Atg5 and Atg7 deletion (4, 5), the role of Unc-51-like kinase 1 (ULK1), a key initiator of autophagy, remains underexplored in in this context. Here, we suggest ULK1 as a critical regulator of pancreatic tumor progression through both cell-intrinsic and immune-modulatory mechanisms. Our findings support a previously unrecognized role for ULK1 in generating a pro-tumorigenic immune microenvironment and suggest that targeting ULK1 may offer a dual therapeutic benefit by impairing autophagy and promoting antitumor immunity.\u003c/p\u003e\n\u003cp\u003eAlthough \u003cem\u003eULK1\u003c/em\u003e mRNA expression is unchanged between normal and tumor patients from human PDAC datasets (TCGA) (Supplementary Fig. S1), our multiplex-IHC and immunoblot analysis revealed that elevated ULK1 activity, as marked by phospho-Atg14 levels (pAtg14), in high-grade human PDAC tissues and cells (Fig. 1), suggesting that ULK1 activity, rather than its expression, is important for tumor progression.\u003c/p\u003e\n\u003cp\u003eThe functional significance of ULK1 in tumor progression is further supported by our findings that ULK1 depletion in both mouse (KPC) and human (MIA PaCa-2) pancreatic cancer cells significantly impaired their proliferation and invasion, supporting its role in tumor aggressiveness (Fig. 2 and Supplementary Fig. S3). Consistent with its crucial role in autophagy activation, ULK1-depleted cells showed defective autophagosome formation, as indicated by reduced LC3-II/I ratios and GFP-LC3 puncta under stress conditions, confirming that ULK1 is critical for maintaining autophagy flux and metabolic adaptation for cancer growth (Fig. 2 and Supplementary Fig. S3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImportantly, our \u003cem\u003ein vivo\u003c/em\u003e studies using both syngeneic orthotopic allografts and spontaneous KPC GEM models provide compelling evidence that Ulk1 is required for pancreatic tumor development in physiological setting. Tumor-intrinsic deletion of \u003cem\u003eUlk1\u003c/em\u003e significantly delayed tumor onset, reduced PDAC burden, and extended survival (Fig. 2, 3\u0026nbsp;and 4). This study is the first\u0026nbsp;direct evidence\u0026nbsp;to demonstrate tumor-promoting function of Ulk1 in a spontaneous PDAC model, suggesting its potential as a promising therapeutic target.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBeyond its canonical role in autophagy-dependent tumor survival, we found an immune-modulatory function of ULK1 in the PDAC tumor microenvironment (TME). Analysis of TCGA datasets revealed a negative correlation between ULK1 expression and MHC class II-mediated antigen presentation pathways (Supplementary Table S5), implicating that ULK1 may suppress antitumor immunity by inhibiting antigen processing in tumor and suppressing antigen presenting cells (APCs) (34, 37).\u0026nbsp;While autophagy has been implicated in immune evasion, particularly through MHC-I degradation of tumor cells\u0026nbsp;(37), our data further revealed that \u003cem\u003eUlk1\u003c/em\u003e-deficient tumors markedly increased the infiltration of MHC II\u003csup\u003e+\u003c/sup\u003e antigen presenting cells (APCs) into tumors, likely potentially enhancing tumor antigen presentation and T cell priming (Fig 5 and Supplementary Fig S5-7).\u003c/p\u003e\n\u003cp\u003eMoreover, genetic deletion of \u003cem\u003eUlk1\u003c/em\u003e in tumor significantly altered the composition of the TME across both orthotopic and spontaneous KPC models. We observed a marked reduction in immunosuppressive polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and neutrophils, accompanied by an increase in CD8⁺ cytotoxic T cells and MHC II⁺ APCs (Fig. 5, 6, and Supplementary Fig. S5-S7).\u0026nbsp;These consistent observations across models strongly suggest that ULK1 contributes to immune evasion by orchestrating the recruitment and maintenance of suppressive myeloid populations while restricting cytotoxic immune cell infiltration.\u003c/p\u003e\n\u003cp\u003eMechanistically, cytokine and chemokine profiling revealed that Ulk1-deficient tumors secreted lower levels of Ccl2, Cxcl2, and G-CSF, key factors that recruit immunosuppressive myeloid cells (43-52). Interestingly, among the cytokines, GM-CSF was the most prominently elevated in \u003cem\u003eUlk1\u003c/em\u003e KO tumors (Fig. 7). Although dual roles of GM-CSF in TME by promoting or inhibiting immune cell subtypes have been reported (53-55), recently GM-CSF shows anti-tumor immune responses by activating M1-macrophages and enhancing DCs differentiation (56-58).\u0026nbsp;The upregulation of DCs populations may explain the increased viability of APCs and further activated T cells\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eobserved in \u003cem\u003eUlk1\u003c/em\u003e-deficient tumors (Fig. 5, 6, Supplementary Fig. S5 and S6). These tumor-intrinsic cytokine changes were recapitulated \u003cem\u003ein vitro\u003c/em\u003e using conditioned media (CM) from \u003cem\u003eUlk1\u003c/em\u003e KO KPC cells, confirming that \u003cem\u003eUlk1\u003c/em\u003e-mediated tumor signaling influences immune cell recruitment through determined by the types of cytokines and chemokines (Fig. 7A-D).\u003c/p\u003e\n\u003cp\u003eFunctional assays further demonstrated that conditioned media from \u003cem\u003eUlk1\u003c/em\u003e KO cells impaired the survival of tumor-infiltrating neutrophils including PMN-MDSCs, while enhancing the viability of CD8⁺ T cells and DCs (Fig. 7E-G and Supplementary Fig. S9A-C). These results suggest that Ulk1-dependent cytokine secretion actively dictates immune cell fate within the tumor niche. These findings highlight the role of Ulk1 in maintaining an immunosuppressive TME, promoting tumor growth not only through autophagy-driven metabolic support but also by modulating immune cell compositions and dynamics.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that autophagy promotes the secretion of pro-tumorigenic factors\u0026mdash;including IL-6, IL-8, and MMP2\u0026mdash;in Ras-driven cancers and facilitates IL-1\u0026beta; secretion through unconventional secretory pathways (59). In line with these studies, our data indicate that ULK1 plays an important role in regulating the secretion of cytokine and chemokines that reprogram the tumor immune landscape. While the precise molecular mechanisms by which ULK1 regulates cytokine expression and trafficking remain to be fully elucidated, our findings strongly implicate ULK1-mediated tumor signals as acting a crucial role in coordinating tumor\u0026ndash;immune interactions through selective modulation of immune-regulatory chemokines and cytokines.\u003c/p\u003e\n\u003cp\u003eIn summary, our study suggests ULK1 as a critical regulator of pancreatic cancer progression through dual mechanisms: sustaining tumor metabolism via autophagy and fostering an immunosuppressive TME by modulating cytokine-mediated immune cell recruitment and survival. These findings support the therapeutic potential of targeting ULK1 in PDAC, offering a strategy to simultaneously impair tumor-intrinsic metabolic support and enhance antitumor immunity.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENT\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank T. Kim (Flow Cytometry Core), K. Kim (Proteomics Core), M. Kim (Microscopy Core), S. Jeon (Molecular Imaging Core), and M. Park (Laboratory Animal Research Core) from Core Center, NCC Korea. The animal study protocols were approved by the Institutional Animal Care and Use Committee of\u0026nbsp;National Cancer Center\u0026nbsp;(Approval #NCC-22-815; #\u0026nbsp;NCC-24-1045)\u0026nbsp;and conducted in accordance with ARRIVE guidelines. This work was supported by the National Cancer Center (NCC-24H1200 and 2510750) and National Research Foundation of Korea (2020R1A2B5B01002011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS \u0026apos; DISCLOSURES\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo disclosures were reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS CONTRIBUTION\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH. Jeong: Conceptualization, formal analysis, validation, investigation, visualization, methodology, writing\u0026ndash;original draft. J. Lee: Conceptualization, formal analysis, validation, investigation, visualization, methodology, writing\u0026ndash;original draft. J. Son: Conceptualization, formal analysis, data curation, investigation, visualization, methodology. J. Lee: Investigation, methodology. M. Kang: Investigation, methodology. S. Cho: Methodology. J.H. Kim: Methodology. Y. Jeon Investigation, Resources, methodology. J. Lee: Data curation, investigation, visualization. D. Shin: Data curation, investigation. supervision, writing\u0026ndash;review and editing. H. Kim: Data curation, supervision, investigation, writing\u0026ndash;review and editing. H. Lee: Resources, supervision, methodology, writing\u0026ndash;review and editing. H. Cheong: Conceptualization, resources, data curation, supervision, funding acquisition, methodology, writing\u0026ndash;original draft, project administration, writing\u0026ndash;review and editing.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHe C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet. 2009;43:67-93.\u003c/li\u003e\n\u003cli\u003eLevine B, Kroemer G. Biological Functions of Autophagy Genes: A Disease Perspective. Cell. 2019;176(1-2):11-42.\u003c/li\u003e\n\u003cli\u003eGuo JY, Chen HY, Mathew R, Fan J, Strohecker AM, Karsli-Uzunbas G, et al. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev. 2011;25(5):460-70.\u003c/li\u003e\n\u003cli\u003eGuo JY, Karsli-Uzunbas G, Mathew R, Aisner SC, Kamphorst JJ, Strohecker AM, et al. Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis. 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Cancer Discov. 2014;4(4):466-79.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6438501/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6438501/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAutophagy plays a dual role in cancer, acting as a tumor suppressor and promoter depending on tumor stage and context. While core autophagy genes such as Atg5 and Atg7, the role of Unc-51-like kinase 1 (ULK1) —a key autophagy initiator-remains poorly understood in pancreatic ductal adenocarcinoma (PDAC). In this study, we investigated the role of ULK1 using tissue-specific deletion in GEM models. Although ULK1 mRNA levels remained unchanged between normal and tumor cells in The Cancer Genome Atlas (TCGA) dataset, multiplex immunohistochemistry revealed elevated ULK1 activity, marked by phosphorylated ATG14, in high-grade human PDAC tissues. Genetic deletion of Ulk1 impaired autophagy, reduced cell proliferation, colony formation, and invasiveness of pancreatic cancer cells. In vivo both syngeneic orthotopic and KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre) mouse model with tissue specific Ulk1 deletion exhibited significant delayed tumor progression, reduced tumor burden, and extended survival.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImportantly, Ulk1 deficiency remodeled the tumor immune microenvironment by reducing tumor-promoting polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and neutrophils while enhancing recruitment of cytotoxic CD8+ T cells and MHC-II+ antigen-presenting cells. Chemokine and cytokine profiling revealed that downregulation of Cxcl2, Ccl2, and G-CSF, might acting for PMN-MDSCs and neutrophils recruitment and survival, with concurrent upregulation of GM-CSF for dendritic cell infiltration, thereby inducing antitumor immunity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese findings provide novel insights into the role of ULK1 in PDAC progression through tumor-intrinsic metabolic support by autophagy activation and immune modulation by tumor-derived cytokines. Targeting ULK1 may represent a promising therapeutic strategy by inhibiting autophagy and enhancing antitumor immune responses in pancreatic cancer.\u003c/p\u003e","manuscriptTitle":"ULK1 Knockout suppresses Pancreatic Cancer Progression by Inhibiting Autophagy and Enhancing Anti-tumor Immunity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 14:41:51","doi":"10.21203/rs.3.rs-6438501/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-06-03T04:55:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-06-01T08:01:15+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-05-26T00:33:35+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-05-14T02:27:48+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-05-13T09:08:53+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-05-09T00:56:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-14T00:05:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-13T09:53:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2025-04-13T09:53:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4a5d1fd1-43c0-4791-8066-8969f434467d","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48281305,"name":"Biological sciences/Cancer/Cancer models"},{"id":48281306,"name":"Biological sciences/Cell biology/Autophagy/Macroautophagy"},{"id":48281307,"name":"Health sciences/Diseases/Cancer/Tumour immunology"}],"tags":[],"updatedAt":"2025-12-18T08:09:51+00:00","versionOfRecord":{"articleIdentity":"rs-6438501","link":"https://doi.org/10.1038/s12276-025-01590-2","journal":{"identity":"experimental-and-molecular-medicine","isVorOnly":false,"title":"Experimental \u0026 Molecular Medicine"},"publishedOn":"2025-12-17 05:00:00","publishedOnDateReadable":"December 17th, 2025"},"versionCreatedAt":"2025-05-13 14:41:51","video":"","vorDoi":"10.1038/s12276-025-01590-2","vorDoiUrl":"https://doi.org/10.1038/s12276-025-01590-2","workflowStages":[]},"version":"v1","identity":"rs-6438501","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6438501","identity":"rs-6438501","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}