Variable Efficacy of the Non-covalent KRAS G12D Inhibitor (MRTX-1133) with Obesity in Murine Pancreatic Cancer

Variable Efficacy of the Non-covalent KRAS G12D Inhibitor (MRTX-1133) with Obesity in Murine Pancreatic Cancer | 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 Short Report Variable Efficacy of the Non-covalent KRAS G12D Inhibitor (MRTX-1133) with Obesity in Murine Pancreatic Cancer Sujith Sarvesh, Holly Stephens, Pia Muri, Henry Nnaemeka Ogbonna, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7871709/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Diet-induced obesity (DIO) promotes pancreatic tumor progression, immunosuppression, and resistance to chemotherapy. The impact of DIO on efficacy of KRAS G12D -specific inhibitor MRTX1133 in syngeneic mouse PDAC cell lines implanted into in lean and DIO mice was evaluated. MRTX1133 induced regression in syngeneic murine PDAC models, irrespective of immune microenvironment composition or body condition, yet, DIO promoted immunosuppression through recruitment of gMDSC into T-cell inflamed TME, reversing control of microscopic disease. Biological sciences/Cancer Health sciences/Oncology Figures Figure 1 Figure 2 Figure 3 Introduction Pancreatic Ductal Adenocarcinoma (PDAC) is currently the third leading cause of cancer-associated mortality in the US 1 with a 5-year survival rates of 13% 1 . Hallmark features of PDAC associated with poor outcomes include mutant KRAS-driven oncogenesis, fibrotic stroma enriched with cancer-associated fibroblasts (CAFs), immunosuppressive, myeloid-derived suppressor cells (MDSCs)/macrophage, and exclusion of cytotoxic T and NK cells from the TME 2 – 3 . Obesity is a major risk factor for PDAC 3 – 5 as it accelerated tumorigenesis, enhances oncogenic KRAS signaling 6 and induces metabolic reprogramming 7 . Diet-induced obesity (DIO) is linked to the development of hyperglycemia, insulin resistance, and type 2 diabetes increasing the risk of developing PDAC 8 . Furthermore, DIO promotes recruitment of immunosuppressive macrophages and MDSCs 8 , 9 , exclusion of T and NK cells and reductions in their cytotoxic activitties 10 – 12 . Targeting oncogenic KRAS signaling has demonstrated potent anti-tumor activity and the ability to reprogram the PDAC TME from a myeloid-enriched, immunosuppressive type to TME enriched with functional cytotoxic T cells and pro-inflammatory macrophage supportive of anti-tumor immunity 13 ,14 15, 16 . Given the impact of obesity on both oncogenic KRAS signaling and tumor immunity, it is of interest to understand the effects of obesity on the efficacy of oncogenic KRAS inhibition in the PDAC TME. As proof of concept, we investigated the efficacy of the Kras G12D inhibitor, MRTX1133, in sucutaneous, syngeneic mouse models of PDAC with different immune contextures in lean and DIO mice. Results and Discussion MRTX1133 inhibits RAS-MAPK signaling, inducing cell cycle arrest and tumor regression As in previous studies 14 , 15 , MRTX1133 treatments in vitro reduced expression of phosphorylated Erk1/2 (pERK1/2), a downstream readout for RAS-MAPK activity (Fig. 1 a-d) and induced G0-G1 phase cell cycle arrest (Fig. 1 e-h) 15 in 2838c3 and 6491c5 PDAC cell lines. Prior to the vivo assessment of MRTX1133 activity, lean and DIO mice were generated (Fig. 2 a, outlined in methods) by feeding diets high or low in fat for a 3-month period induce mice with lean (mean = 22.58 gm) vs obese (mean = 39.65 gm) body masses (Fig. 2 b). Secondary alterations in fasted blood glucose values (Fig. 2 c) were noted, consistent with DIO-induced pre-diabetic state 17 . After generation of lean and DIO C57BL/6 mice, mice were randomized into groups and implanted with tumors. When 2838c3 and 6419c5 tumors reached 50–70 mm 3 , lean and DIO mice were treated with MRTX1133 (Fig. 2 d; outlined in methods). MRTX1133 induced regression of established 2838c3 tumors in both lean and DIO mice (Fig. 2 e-f) and relapsed post-MRTX1133 treatment in 27% of lean (3/9 mice) and 89% (8/9 mice) DIO 2838c3 tumor bearing mice (Fig. 2 g), implying a reduction in control of microscopic disease in DIO model. In comparison, T cell-excluded, 6419c5 tumors responded to MRTX1133 similarly irrespective of body mass (Fig. 2 h-i). Upon termination of MRTX1133 treatment, 6419c5 tumors regrew in lean and DIO (Fig. 2 j) mice, indicating no control of microscopic disease. Overall, the responses of each cell line to MRTX1133 were similar to previous findings in lean body mass mice 15 and indicated that DIO did not affect the in vivo cytotoxic activity of MRTX133, which is primarily attributed to inhibition of S-phase 14 , 15 whereas, control of microscopic disease in 2838c3, attributed to intratumoral T cell activity, was inhibited in DIO mice. DIO increases granulocytic MDSCs (gMDSCs) infiltration into the MRTX1133-treated, 2838c3 TME Recurrence of tumors in the DIO 2838c3 model suggested a role for obesity-induced suppression of T cell control of microscopic disease 15 or an impact on MRTX1133-induced cytotoxic T cell activity 16 . Host obesity alters the immune composition of the TME, increasing recruitment of immunosuppressive gMDSCand excluding cytotoxic CD8 + T cells 18 . Therefore, we evaluated changes in immune cell infiltration using multiparameter flow cytometry and NanoString mRNA profiling, focusing on myeloid and T cell subsets in lean versus DIO mice with established 2838c3 tumors after completion of MRTX1133 treatment. To do this, we adjusted the dosing schedule to ensure sufficient 2838c3 tumor tissue (cells and mRNA) prior to evaluation of changes in immune cell recruitment and function ( Supplementary Fig. 1a ; outlined in methods). Tumors in both models responded robustly to the modified MRTX133 treatment regimen ( Supplementary Fig. 1b ) indicating efficacy in larger tumors (TV = 250-500mm 3 ). Consistent gross evidence of regression, CD45 − YFP + tumor cells were reduced in MRTX1133 treated LFD and HFD cohorts ( Supplementary Fig. 1c-d ) and mRNAs associated with cell cycle progression were reduced with MRTX1133 treatment ( Supplementary Fig. 1e ) supporting observation of S-phase inhibition in vivo 15 in both lean and DIO models. Evaluation of myeloid cell infiltration in the 2838c3 TME, revealed a significant increase in proportions of Ly6C int Ly6G + gMDSC (Fig. 3 a) and increased expression of PD-L1 and CD206 on gMDSC within 2838c3 DIO tumors after MRTX1133 treatment (Fig. 3 b). No significant changes were noted in abundances of CD11b + Ly6G/C − F4/80 + macrophages (Fig. 3 c), however, an increase in proportions of MHCII + CD206 − pro-inflammatory M1 like macrophage in lean 2838c3 tumors and a decrease in MHCII −/+ CD206 + anti-inflammatory M2 like macrophage was noted with MRTX1133 treatment in both lean and DIO tumors (Fig. 3 d). In comparison to 2838c3, minimal differences in abundance of gMDSCs (already elevated) and F4/80 + macrophage were observed in 6419c5 (both lean and DIO) with MRTX1133 treatment ( Supplementary Fig. 2a-d ). However, similar to 2838c3, MRTX1133 treatment induced M1 macrophage polarization ( Supplementary Fig. 2e ) implying similar impact of MRTX1133 inhibition of oncogenic Kras G12D signaling on macrophage polarization in both models 15 . To better understand the impact of DIO on innate cell changes in the DIO 2838c3 TME, we performed analysis of differential gene expression in 2838c3 lean and DIO models treated with MRTX1133 (Fig. 3 e). Significant increases in CD36 (Fig. 3 e-f) and mRNAs ANGPTL4, COL11A1, and SERPINB5, which are involved directly in promoting tumor proliferation, migration, metastasis 19 – 21 and EMT 22 , were noted. Interestingly, CD36 is expressed on a wider range of cells within tumor microenvironment including tumor, stromal, regulatory T cells, CAFs and immunosuppressive myeloid cells 23 and elevated CD36 mRNA in PDAC patients is associated with poor outcomes (Fig. 3 g) 24 – 25 . Previous studies support a role for CD36 in recruitment, metabolic reprogramming, and immunosuppressive activities of gMDSC 26 – 27 suggesting that DIO impacts gMDSC recruitment into the 2838c3 TME and may involve CD36 signaling. Overall, these findings suggest potential tumor-, stromal-, and immune-specific adaptations in DIO mice in response to MRTX1133 that have the potential to impact treatment outcomes. DIO reduces T cell infiltration and cytotoxic effector activity of T cells post MRTX1133 treatment Previous work indicates a correlation between increasing gMDSC infiltration into PDAC TME and a reduction CD8 + T cell abundance and function 18 . Therefore, we evaluated the impact of DIO on the T cells response to MRTX1133 treatment within the TiME of 2838c3 tumors. Analysis of CD3 + T cell infiltrate within TiME revealed reductions in CD4 + Foxp3 − (Fig. 3 h) and CD8 + T cells (Fig. 3 i) in MRTX1133-treated, 2838c3 tumors from DIO mice. Furthermore, the ratio of CD8 + T cells to gMDSC was significantly reduced in MRTX1133-treated 2838c3 tumors from DIO mice (Fig. 3 j) suggesting an important imbalance in cytotoxic T cells to gMDSC abundances. Analysis of T cell activation (PD-1, CD69, CD44, CD62L) and proliferation (Ki67) revealed significant increases in PD-1 ( Supplementary Fig. 3a ), CD69 ( Supplementary Fig. 3b ) , trending increase of CD44 hi CD62L − ( Supplementary Fig. 3c ) abundance and increases in Ki67 + proliferation ( Supplementary Fig. 3d ) within CD4 + and CD8 + T cells supporting a shift to effector T cell phenotype. Furthermore, CD8 + and CD4 + T cells isolated from MRTX1133-treated, 2838c3 TME from DIO mice produced less IFNγ (Fig. 3 k) after in vitro stimulation. These findings are consistent with hypothesis that increased gMDSC infiltration into the TME impacts T cell infiltration and function in the TiME and most likely turnover 28 resulting in reductions in T-cell mediated control of microscopic tumor growth. Analysis of mRNA revealed significant downregulation of mRNA associated with T cell abundances, trafficking, retention, and Th1 effector activities including CD3d, CD3g, Foxp3, CXCR3, CXCR6, NKG7, LTB, and DPP4 (Fig. 3 e). Pathway and gene ontology analysis revealed downregulation of pathways associated with positive regulation of leukocyte activation, migration, and NK cell immunity ( Supplementary Fig. 3e ). In comparison to 2838c3 tumors, no significant differences were noted in T or NK cell abundance ( Supplementary Fig. 4a-bB) , CD8: gMDSC ratios ( Supplementary 4c ), T cell expression of PD-1, CD69, Ki67 ( Supplementary Fig. 4d-f ) and IFNγ ( Supplementary Fig. 4g ) in MRTX1133 treatment in 6419c5 lean and DIO cohorts. Overall, these findings indicate a role for obesity driven inflammation in dampening T cell-mediated control of residual microscopic disease after targeted KRAS G12D inhibition that may negatively impact efficacy of parenteral administration of MRTX11333 in obese patients, resulting in greater rates of disease recurrence. Additional studies on the molecular machinery involved, in particular CD36 and ANGPTL4, as promoters of metabolic reprogramming of cancer and immune cells, SERPINB5 and COL11A1, as modulators of stromal responses to treatment and tumor progression, and the impact of MDSC depletion and PD-1 ICB in combination with MRTX1133 in obese models of PDAC is warranted. Declarations Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed by the authors. Authors’ Contributions: Conception and design: Lyse A. Norian and Jeremy B. Foote Development of methodology: Lyse A. Norian and Jeremy B. Foote Acquisition of data: Sujith Sarvesh, Jeremy B. Foote, Cherlene Hardy, Pia Muri, Henry Nnaemeka Ogbonna, Lyse A. Norian Analysis and interpretation of data: Sujith Sarvesh, Holly Stephens, Jianqing Zhang, Lyse A. Norian and Jeremy B. Foote Writing, review, and/or editing of manuscript: Sujith Sarvesh, Dhana Sekar Reddy Bandi, Bassel F. El-Rayes, Lyse A. Norian, and Jeremy B. Foote Administrative, technical, or material support: Bassel F. El-Rayes, Ganji Purnachandra Nagaraju, Lyse A. Norian, and Jeremy B. Foote Study Supervision: Lyse A. Norian and Jeremy Foote Acknowledgements: Research histology services were provided by the Comparative Pathology Laboratory (Animal Resources Program), NanoString by UAB NanoString Laboratory (Department of Radiation Oncology) and Flow Cytometry support by the UAB Flow Cytometry Core (S10 OD032296). Funding: The research was funded by an intramural 2022-2023 pilot grant award to Lyse A. Norian and Jeremy B. Foote from the UAB Departments of Microbiology and the Nutrition Obesity Research Center (NORC) for the grant proposal application “Targeted Inhibition of Oncogenic KRAS in Lean and Obese Pancreatic Ductal Adenocarcinoma Tumor Immune Microenivornment”. Materials and Methods Animals Eight-week-old female C57BL/6 mice (000664) mice were purchased from Jackson Laboratory and used for these studies to reduce incidence of aggression and injury seen in male mice when fed high fat diets. All animal experiments and procedures were performed in accordance with guidelines from the University of Alabama at Birmingham’s Institutional Animal Care & Use Committee (IACUC) using an approved IACUC protocol (APN22661). Diet Following one week of acclimation, animals were randomized to one of two matched, purified-ingredient diets: a low-fat diet (LFD; Research Diets 12450J with 10% kcal from fat and matching sucrose to 12492) or high-fat diet (HFD; Research Diets 12492 with 60% kcal from fat) for 12-14 weeks, thereby generating lean (LFD) or diet-induced obese (DIO, HFD) mice. Mice were weighed weekly and fasted for 24 hours followed by an assessment of blood glucose levels. Prior to use, individual mice were identified as DIO if their final body weight was > 3 s.d. above the mean of age-matched lean (LFD-fed) mice within the same cohort, as per our reported methods 32 . Before tumor cell implantation and initiation of in vivo studies, we established mice with the appropriate body conditions by feeding either low fat diet (LFD; 10% kcal from fat) or high fat diet (HFD, 60% kcal from fat) for 12 weeks 28 . After this, mice were weighed and classified as either lean (LFD, final body weight 3 s.d. above the LFD mean) based on established metrics 18,28 Cell lines Murine PDAC cell lines purchased from Kerafast (Boston, MA) were derived by Li et al. 21 from tumor-bearing Kras LSL-G12D/+ ; Trp53 LSL-R172H/+ ; Pdx1-Cre; Rosa26 YFP/YFP (6419c5, 2838c3) mice and cultured in DMEM complete media as previously indicated 29 . Tumor cell implantation A single-cell suspension of murine PDAC cells was prepared in PBS and kept on ice until injection. Tumor cells (1 × 10 6 ) were injected subcutaneously into the flank regions of 8-week-old, female C57BL/6 mice. MRTX1133 formulation For in vitro treatments, MRTX1133 (Chemitek, Indianapolis IN) was dissolved in DMSO at a concentration of 25 μM and diluted to 10, 100, 200, 500, 1000 ηM concentrations in DMEM complete media. PDAC tumor cell lines (2838c3 and 6419c5) were plated at 1.5 x 10 5 cells/well in 6-well plates and incubated in working concentrations of MRTX1133 above for either 3 or 24 hours. Cells were then washed 3x in PBS and lysates were generated using RIPA buffer with protease and phosphatase inhibitors for downstream immunoblotting. For in vivo studies MRTX1133 was formulated in 10% research grade Captisol (CyDex Pharmaceuticals) in 50 mmol/L citrate buffer pH 5.0. Once formulated, MRTX1133 was protected from light and stored at 4°C for 1 week treatment course. MRTX1133 was administered at 30 mg/kg via i.p. injection with b.i.d. dosing either daily (tumor growth curves) after tumors reached an average volume of 50-70 mm 3 or in a modified dosing schedule (1 day SID, 3 days BID, 1 day rest, 1 day SID at endpoint) in order to preserve tumor for downstream flow cytometry profiling of the immune microenvironment. After terminating treatment with MRTX1133 at day 8, MRTX1133-treated 2838c3 lean and DIO cohorts were monitored for 60 days using bi-weekly measurements of tumors. Mice with tumor implantation sites containing no palpable evidence of tumor mass (< 3 x 3 mm) were considered to lack gross tumor burden (complete regression). Prior to determining the impact of DIO on MDSC recruitment, we adjusted the dosing schedule to ensure sufficient 2838c3 tumor tissue was present for downstream analysis after termination of MRTX1133 treatment. The following changes to the MRTX1133 treatment regimen were used with YFP-expressing 2838c3 and 6419c5 cells: (i) tumors were grown for a longer period of time (~14 days) to generate larger tumors (~ 300 – 700 mm 3 ) prior to treatment and (ii) the treatment schedule was abbreviated to an initial PM dose on day 1, AM/PM dosing on days 2-4, a day of rest on day 5, followed by an AM dose on day 6, exactly 3 hours prior to tissue collection for FACS, NanoString, and histology. NanoString and Gene Ontology Analysis of Tumor Immune Microenvironment At day 20 experimental endpoint, 2838ce and 6419c5 PDAC tumors were excised and a portion was preserved in RNAlater. RNA was batch isolated and submitted to the UAB NanoString facility for interrogation using the nCounter PanCancer Immune Profiling Panel (XT-CSO-MIP1-12) according to the manufacturer’s instructions. Data analysis was performed using the Rosalind Bioinformatics Analysis Platform for NanoString nCounter Gene Analysis. Differentially expressed genes were identified as those with a p value ± 1.5. Due to the exploratory nature of our study and intent for validation, we used raw p values and did not adjust p values for multiple comparisons. The NanoString tumor immunogenetic profiling dataset expression matrix was composed of n=12 mice bearing 2838c3 tumors consisting of the following groups: (i) vehicle treated, lean body mass, (ii) MRTX1133 treated, lean body mass, (iii) vehicle treated, DIO, and (iv) MRTX1133 treated, DIO mice. Differentially expressed genes were separated into groups of upregulated or downregulated genes and subjected to Gene Ontology enrichment analysis using the PANTHER classification system to identify associated biological processes (http://geneontology.org/). Immunoblotting Tumor or cell lysates were generated after treatment with RIPA lysis buffer (Thermoscientific, IL, USA) with protease and phosphatase inhibitors (Milipore, MA, USA) and sonicated for homogenization and equal protein concentrations were loaded and run on a 4-20% SDS-PAGE. Gels were transferred to a PVDF membrane, which were blocked with or 5% BSA in TBS-T (TBS with 0.1% Tween 20) depending on the blocking agent used for the primary antibodies, for 1 hour at room temperature. Membranes were incubated overnight at 4°C on a rocker with primary antibodies ( Supplementary Table 1 ) according to the manufacturer’s instructions and then incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology) for 45 min at room temperature on an orbital shaker. Membranes were then washed to remove excess secondary antibody and developed using SuperSignal West Enhanced Chemiluminescent substrate (Thermo Fisher Scientific) and imaged using a Odyssey XF (Li-Cor). Representative whole blots (pERK, tERK, βactin) for 2838c3 ( Supplementary Figure 5 ) and 6419c5 ( Supplementary Figure 6 ) at 3 hours post incubation in vehicle/MRTX1133 are included. Multiparameter Flow Cytometry Mouse tumors were digested in a 1 mg/mL collagenase IV and 0.1 mg/mL DNAase 1 (Worthington Biochemical, Lakewood, NJ) in HBSS for 45 minutes at 37 °C with intermittent shaking. Samples were then washed with RPMI containing 10% FBS and filtered through a 70μM strainer generating single cell suspensions. Cells were labeled with primary fluorophore-conjugated antibodies and a live/dead stain ( Supplementary Table 2 ) for 30-60 min at 4 °C, washed and re-suspended in flow buffer(2% Fetal bovine serum in DPBS). Cytokine expression from single cell suspensions of spleen and tumor was quantified as follows: cells were plated at a concentration of 10 6 cells in 1 mL of RP-10(RPMI + 10% FBS) media and stimulated for 5 hours at 37 °C using a 1x solution of a Cell Activation Cocktail containing PMA ionomycin, and Brefeldin A (Biolegend, San Diego, CA). Conversely, cells were cultured for 5 hours at 37°C using a solution containing 5 ug of Brefeldin A (BioLegend, San Diego, CA) to serve as an unstimulated control. After cell surface staining, cells were fixed in 4% paraformaldehyde for 45 minutes at 4 °C then washed with 1x Perm/Wash (BD, 554723). For intracellular staining in tumor and spleen samples, cells were stained with an antibody cocktail specific intracellular cytokines (IL-2, IFNγ, TNFα, IL-17A, and granzyme B) in Perm/Wash solution for 60 minutes at 4 °C. Data were acquired on a Symphony A5 flow cytometer, with analysis performed using FlowJo version 10.7.2. Statistical Analyses GraphPad Prism (version 9.1.2) was used for statistical analyses and graphical representation with data are presented as either means ± standard deviation (SD) or standard error of the mean (SEM). Two-tailed Student’s t-test and Two-way Analysis of Variance (ANOVA) with multiple corrections (Bonnferoni method) were performed for determination of statistical significance between groups. References Siegel RL, Kratzer TB, Giaquinto AN, Sung H, Jemal A. Cancer statistics, 2025. Ca . 2025;75(1):10. Karamitopoulou E. Tumour microenvironment of pancreatic cancer: immune landscape is dictated by molecular and histopathological features. Br J Cancer . Jul 2019;121(1):5-14. doi:10.1038/s41416-019-0479-5 Xu M, Jung X, Hines OJ, Eibl G, Chen Y. Obesity and pancreatic cancer: overview of epidemiology and potential prevention by weight loss. Pancreas . 2018;47(2):158-162. Rawla P, Thandra KC, Sunkara T. 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Supplementary Files npjPrecisionOncologySupplementaryFiguresandLegends.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 17 Nov, 2025 Reviews received at journal 11 Nov, 2025 Reviewers agreed at journal 01 Nov, 2025 Reviews received at journal 29 Oct, 2025 Reviews received at journal 28 Oct, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviewers invited by journal 27 Oct, 2025 Editor assigned by journal 26 Oct, 2025 Submission checks completed at journal 26 Oct, 2025 First submitted to journal 15 Oct, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7871709","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":538501317,"identity":"3db2b0b5-62c3-4c19-ae05-9dab67b6eb28","order_by":0,"name":"Sujith Sarvesh","email":"","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Sujith","middleName":"","lastName":"Sarvesh","suffix":""},{"id":538501319,"identity":"64394410-2431-41b6-b3ec-a992b652cd0b","order_by":1,"name":"Holly Stephens","email":"","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Holly","middleName":"","lastName":"Stephens","suffix":""},{"id":538501327,"identity":"98627c6d-cfd1-4de8-ad2f-f0bbb37f21ef","order_by":2,"name":"Pia Muri","email":"","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Pia","middleName":"","lastName":"Muri","suffix":""},{"id":538501328,"identity":"34ac5d50-5b6e-4d10-8b52-2d40f79841bc","order_by":3,"name":"Henry Nnaemeka Ogbonna","email":"","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Henry","middleName":"Nnaemeka","lastName":"Ogbonna","suffix":""},{"id":538501329,"identity":"1b49019a-43d2-479f-ac22-49b0af450188","order_by":4,"name":"Jianqing Zhang","email":"","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Jianqing","middleName":"","lastName":"Zhang","suffix":""},{"id":538501331,"identity":"7e17bd62-5e75-453a-a1f1-d451787382f9","order_by":5,"name":"Dhana Sekar Reddy Bandi","email":"","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Dhana","middleName":"Sekar Reddy","lastName":"Bandi","suffix":""},{"id":538501332,"identity":"f8c76c09-521a-413e-9396-53ecc3a9fafd","order_by":6,"name":"Cherlene Hardy","email":"","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Cherlene","middleName":"","lastName":"Hardy","suffix":""},{"id":538501333,"identity":"4c544d1f-b478-4ab2-92b6-7176ecb8b0bc","order_by":7,"name":"Ganji Purnachandra Nagaraju","email":"","orcid":"","institution":"University of Alabama at Birmingham","correspondingAuthor":false,"prefix":"","firstName":"Ganji","middleName":"Purnachandra","lastName":"Nagaraju","suffix":""},{"id":538501334,"identity":"28d7d89d-f798-4f91-9eaf-75857ac9a1dc","order_by":8,"name":"Bassel F. 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09:29:52","extension":"xml","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90120,"visible":true,"origin":"","legend":"","description":"","filename":"746b663869f64dd9ab36c051113a4aa81structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7871709/v1/49e2e578c083d7f46efebeea.xml"},{"id":95282786,"identity":"38fc91b2-a603-4ec6-a182-2870ca915834","added_by":"auto","created_at":"2025-11-06 09:29:48","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102039,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7871709/v1/748b9c0a07ae6fab8a4b481e.html"},{"id":95282794,"identity":"32511723-e7d5-443c-beba-fc2fdb928b88","added_by":"auto","created_at":"2025-11-06 09:29:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":292314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eMRTX1133 inhibits RAS-MAPK signaling, inducing cell cycle arrest and growth of syngeneic mouse PDAC cell lines in vitro.\u003c/u\u003e\u003cstrong\u003e a-b) \u003c/strong\u003eImmunoblotting of phosphorylated-ERK (pERK), ERK, β-Actin as loading control and quantification of pERK levels normalized to β-Actin following treatment of syngeneic mouse PDAC cell line 2838c3 \u003cem\u003ein vitro\u003c/em\u003e with 10, 100, 200, 500 and 1000 nM of MRTX1133 at 3 hrs. \u003cstrong\u003ec-d) \u003c/strong\u003eImmunoblotting of pERK, ERK, β-Actin as loading control and quantification of pERK levels normalized to β-Actin following treatment of syngeneic mouse PDAC cell line 6419c5 \u003cem\u003ein vitro\u003c/em\u003e with 10, 100, 200, 500 and 1000 nM of MRTX1133 at 3 hrs. \u003cstrong\u003ee)\u003c/strong\u003e Histogram representing different phases of cell cycle in a propidium iodide based flow cytometry assay of 2838c3 following treatment with MRTX1133 at 200nm concentration\u003cstrong\u003e f) \u003c/strong\u003eQuantification of cell cycle phases of 2838c3 PDAC cell line in vitro at 100,500 and 1000nM concentrations of MRTX1133. \u003cstrong\u003eg) \u003c/strong\u003eHistogram representing different phases of cell cycle in a propidium iodide-based flow cytometry assay of 6419c5 following treatment with MRTX1133 at 200nm concentration\u003cstrong\u003e h)\u003c/strong\u003eQuantification of cell cycle phases of 6419c5 PDAC cell line in vitro at 100,500 and 1000nM concentrations of MRTX1133. All data represented as Mean ± SD.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7871709/v1/e31eeebeca2bd0aff93d7522.png"},{"id":95314265,"identity":"9264c2c3-9f2e-47e5-8354-d05824fb9eb3","added_by":"auto","created_at":"2025-11-06 15:52:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":818019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eMRTX1133 induces tumor regression in both the 2838c3 and 6419c5 murine models of PDAC. \u003c/u\u003e\u003cstrong\u003ea)\u003c/strong\u003e Generation of Diet-Induced Obese and Low-Fat diet induced murine models. \u003cstrong\u003eb) \u003c/strong\u003eBody weights of mice in low-fat diet and high-fat diet cohorts, mean +/- SD (n = 15-20 mice/diet). Student’s t test (**** p \u0026lt;0.0001). \u003cstrong\u003ec)\u003c/strong\u003e Blood glucose levels of the mice in the low-fat diet and high-fat diet cohort recorded at the end of 12 weeks low-fat/high-fat diet program, mean +/- SD, n = 15-20 mice/diet. Student’s t test (**p\u0026lt;0.01). \u003cstrong\u003ed)\u003c/strong\u003e Experimental design where the Low-fat diet and DIO mice are implanted with syngeneic PDAC cell lines (2838c3 and 6419c5) subcutaneously and subsequently treated with either vehicle or MRTX1133 (30mg/kg bid for 7 days) and were euthanized at ~ day 67 for 2838c3 and ~ day 30 for 6419c5 based on tumor regrowth. \u003cstrong\u003ee)\u003c/strong\u003e Tumor volume (in mm\u003csup\u003e3\u003c/sup\u003e) changes over the course of experiment in 2838c3 tumor bearing mice, mean +/- SD, n = 7 – 8 mice/group, representative of two independent experiments. \u003cstrong\u003ef) \u003c/strong\u003ePercent change in tumor volumes between vehicle and MRTX1133 in LFD and HFD cohort of 2838c3 tumor bearing mice, mean +/- SD, n = 7-8 mice/group, two-way ANOVA with Sidak multiple comparisons (**p\u0026lt;0.001 and ****p\u0026lt;0.0001). \u003cstrong\u003eg) \u003c/strong\u003ePercentage of regrowth of 2838c3 tumors after regressing within the treatment window (7 days) in LFD and HFD cohorts. \u003cstrong\u003eh)\u003c/strong\u003e Tumor volume (in mm\u003csup\u003e3\u003c/sup\u003e) changes over the course of experiment in 6419c5 tumor bearing mice, mean +/- SD with 5 – 8 mice/group representative of two independent experiments. \u003cstrong\u003ei) \u003c/strong\u003ePercent change in tumor volumes between vehicle and MRTX1133 in LFD and HFD cohort of 2838c3 tumor bearing mice, n = 5-8 mice/group, two-way ANOVA with Sidak multiple comparisons (*p\u0026lt;0.05 and **p\u0026lt;0.01). \u003cstrong\u003ej)\u003c/strong\u003e Percentage of regrowth of 6419c5 tumors after regressing within the treatment window (7 days) in LFD and HFD cohorts.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7871709/v1/46124997ff858b679f620bb4.png"},{"id":95282789,"identity":"5b8943f6-1fb0-4e48-a1cf-43a3668dd212","added_by":"auto","created_at":"2025-11-06 09:29:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":871548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eDiet-induced obesity alters impact of MRTX1133 induced KRAS inhibition on the tumor immune microenvironment.\u003c/u\u003e\u003cstrong\u003e a) \u003c/strong\u003e% CD45\u003csup\u003e+\u003c/sup\u003e that are CD11b\u003csup\u003e+\u003c/sup\u003e Ly6G\u003csup\u003e+\u003c/sup\u003e gMDSC within TME in LFD and DIO post vehicle and MRTX1133 treatment. \u003cstrong\u003eb)\u003c/strong\u003e PD-L1 and CD206 expression in gMDSC within the TME in LFD and DIO post vehicle and MRTX1133 treatment. \u003cstrong\u003ec) \u003c/strong\u003e%CD45\u003csup\u003e+\u003c/sup\u003e that are CD11b\u003csup\u003e+\u003c/sup\u003e Ly6C/G\u003csup\u003e-\u003c/sup\u003e F4/80\u003csup\u003e+\u003c/sup\u003e macrophage within TME in LFD and DIO post vehicle and MRTX1133 treatment. \u003cstrong\u003ed)\u003c/strong\u003e Frequencies and ratio of M1 (MHCII\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e-\u003c/sup\u003e) and M2 (MHCII\u003csup\u003e-\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003e) macrophage within the in LFD and DIO post vehicle and MRTX1133 treatment. \u003cstrong\u003ee) \u003c/strong\u003eCD36 mRNA expression within TME in LFD and DIO post vehicle and MRTX1133 treatment, n = 3/group, one-way ANOVA with Šidák multiple comparisons (*p\u0026lt;0.05). \u003cstrong\u003ef) \u003c/strong\u003eSurvival of PDAC patients with high (n = 44) vs. medium to low (n = 142) CD36 mRNA expression as calculated from patient mRNA TCGA data using UALCAN. Statistical significance was calculated using Log Rank test. \u003cstrong\u003eg) \u003c/strong\u003e%CD45\u003csup\u003e+\u003c/sup\u003e of CD4 Foxp3- and CD4+ Foxp3+ T cells within TME in LFD and DIO post vehicle and MRTX1133 treatment. \u003cstrong\u003eh) \u003c/strong\u003e%CD45\u003csup\u003e+ \u003c/sup\u003eof CD4\u003csup\u003e+\u003c/sup\u003e Foxp3\u003csup\u003e-\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e Foxp3\u003csup\u003e+\u003c/sup\u003e T cells within TME in LFD and DIO post vehicle and MRTX1133 treatment. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003cstrong\u003ei) \u003c/strong\u003e%CD45\u003csup\u003e+ \u003c/sup\u003eof CD8\u003csup\u003e+\u003c/sup\u003e T cells within TME in LFD and DIO post vehicle and MRTX1133 treatment. \u003cstrong\u003ej) \u003c/strong\u003eRatio of CD8\u003csup\u003e+ \u003c/sup\u003eT cells to gMDSCs in vehicle and MRTX1133 treated cohort of LFD and HFD 2838c3 tumor bearing mice. \u003cstrong\u003ek) \u003c/strong\u003eCytoplasmic expression of IFNγ was evaluated in CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cells after a 5-hour stimulation with PMA and ionomycin in presence of brefeldin A.\u003cstrong\u003e \u003c/strong\u003eFor all FACS data (with exception of MFI data-which is representative of 2 independent experiments) in panels \u003cstrong\u003ea-k)\u003c/strong\u003e is represented as an aggregate of two independent experiments with n = 8 – 14 mice/group, +/- SEM. Statistical significance across groups was calculated using two-way ANOVA with Tukey’s multiple comparison test to correct for type 1 error. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7871709/v1/991b305c773acbee0278aaef.png"},{"id":95523750,"identity":"dccf068a-1247-47e0-829c-f9bd2ef7790f","added_by":"auto","created_at":"2025-11-10 10:00:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2875111,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7871709/v1/b3c03269-9e5c-454b-80db-5d8589021ebb.pdf"},{"id":95314007,"identity":"3bbc7334-f543-4d69-82fa-8253f3d2067a","added_by":"auto","created_at":"2025-11-06 15:52:22","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1494257,"visible":true,"origin":"","legend":"","description":"","filename":"npjPrecisionOncologySupplementaryFiguresandLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-7871709/v1/49e8f56bade32ba323341d81.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Variable Efficacy of the Non-covalent KRAS G12D Inhibitor (MRTX-1133) with Obesity in Murine Pancreatic Cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic Ductal Adenocarcinoma (PDAC) is currently the third leading cause of cancer-associated mortality in the US\u003csup\u003e1\u003c/sup\u003e with a 5-year survival rates of 13%\u003csup\u003e1\u003c/sup\u003e. Hallmark features of PDAC associated with poor outcomes include mutant KRAS-driven oncogenesis, fibrotic stroma enriched with cancer-associated fibroblasts (CAFs), immunosuppressive, myeloid-derived suppressor cells (MDSCs)/macrophage, and exclusion of cytotoxic T and NK cells from the TME\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eObesity is a major risk factor for PDAC\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e as it accelerated tumorigenesis, enhances oncogenic KRAS signaling\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and induces metabolic reprogramming\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Diet-induced obesity (DIO) is linked to the development of hyperglycemia, insulin resistance, and type 2 diabetes increasing the risk of developing PDAC\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Furthermore, DIO promotes recruitment of immunosuppressive macrophages and MDSCs\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, exclusion of T and NK cells and reductions in their cytotoxic activitties\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTargeting oncogenic KRAS signaling has demonstrated potent anti-tumor activity and the ability to reprogram the PDAC TME from a myeloid-enriched, immunosuppressive type to TME enriched with functional cytotoxic T cells and pro-inflammatory macrophage supportive of anti-tumor immunity\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,14 15,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Given the impact of obesity on both oncogenic KRAS signaling and tumor immunity, it is of interest to understand the effects of obesity on the efficacy of oncogenic KRAS inhibition in the PDAC TME.\u003c/p\u003e\u003cp\u003eAs proof of concept, we investigated the efficacy of the Kras\u003csup\u003eG12D\u003c/sup\u003e inhibitor, MRTX1133, in sucutaneous, syngeneic mouse models of PDAC with different immune contextures in lean and DIO mice.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMRTX1133 inhibits RAS-MAPK signaling, inducing cell cycle arrest and tumor regression\u003c/h2\u003e\u003cp\u003eAs in previous studies\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, MRTX1133 treatments \u003cem\u003ein vitro\u003c/em\u003e reduced expression of phosphorylated Erk1/2 (pERK1/2), a downstream readout for RAS-MAPK activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-d) and induced G0-G1 phase cell cycle arrest (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-h)\u003csup\u003e15\u003c/sup\u003e in 2838c3 and 6491c5 PDAC cell lines. Prior to \u003cem\u003ethe vivo\u003c/em\u003e assessment of MRTX1133 activity, lean and DIO mice were generated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, outlined in methods) by feeding diets high or low in fat for a 3-month period induce mice with lean (mean\u0026thinsp;=\u0026thinsp;22.58 gm) vs obese (mean\u0026thinsp;=\u0026thinsp;39.65 gm) body masses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Secondary alterations in fasted blood glucose values (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) were noted, consistent with DIO-induced pre-diabetic state\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. After generation of lean and DIO C57BL/6 mice, mice were randomized into groups and implanted with tumors. When 2838c3 and 6419c5 tumors reached 50\u0026ndash;70 mm\u003csup\u003e3\u003c/sup\u003e, lean and DIO mice were treated with MRTX1133 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed; outlined in methods). MRTX1133 induced regression of established 2838c3 tumors in both lean and DIO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-f) and relapsed post-MRTX1133 treatment in 27% of lean (3/9 mice) and 89% (8/9 mice) DIO 2838c3 tumor bearing mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg), implying a reduction in control of microscopic disease in DIO model. In comparison, T cell-excluded, 6419c5 tumors responded to MRTX1133 similarly irrespective of body mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh-i). Upon termination of MRTX1133 treatment, 6419c5 tumors regrew in lean and DIO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej) mice, indicating no control of microscopic disease.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOverall, the responses of each cell line to MRTX1133 were similar to previous findings in lean body mass mice\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and indicated that DIO did not affect the \u003cem\u003ein vivo\u003c/em\u003e cytotoxic activity of MRTX133, which is primarily attributed to inhibition of S-phase\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e whereas, control of microscopic disease in 2838c3, attributed to intratumoral T cell activity, was inhibited in DIO mice.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDIO increases granulocytic MDSCs (gMDSCs) infiltration into the MRTX1133-treated, 2838c3 TME\u003c/h3\u003e\n\u003cp\u003eRecurrence of tumors in the DIO 2838c3 model suggested a role for obesity-induced suppression of T cell control of microscopic disease\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e or an impact on MRTX1133-induced cytotoxic T cell activity\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Host obesity alters the immune composition of the TME, increasing recruitment of immunosuppressive gMDSCand excluding cytotoxic CD8\u003csup\u003e+\u003c/sup\u003e T cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTherefore, we evaluated changes in immune cell infiltration using multiparameter flow cytometry and NanoString mRNA profiling, focusing on myeloid and T cell subsets in lean versus DIO mice with established 2838c3 tumors after completion of MRTX1133 treatment. To do this, we adjusted the dosing schedule to ensure sufficient 2838c3 tumor tissue (cells and mRNA) prior to evaluation of changes in immune cell recruitment and function (\u003cb\u003eSupplementary Fig.\u0026nbsp;1a\u003c/b\u003e; outlined in methods). Tumors in both models responded robustly to the modified MRTX133 treatment regimen (\u003cb\u003eSupplementary Fig.\u0026nbsp;1b\u003c/b\u003e) indicating efficacy in larger tumors (TV\u0026thinsp;=\u0026thinsp;250-500mm\u003csup\u003e3\u003c/sup\u003e). Consistent gross evidence of regression, CD45\u003csup\u003e\u0026minus;\u003c/sup\u003e YFP\u003csup\u003e+\u003c/sup\u003e tumor cells were reduced in MRTX1133 treated LFD and HFD cohorts (\u003cb\u003eSupplementary Fig.\u0026nbsp;1c-d\u003c/b\u003e) and mRNAs associated with cell cycle progression were reduced with MRTX1133 treatment (\u003cb\u003eSupplementary Fig.\u0026nbsp;1e\u003c/b\u003e) supporting observation of S-phase inhibition \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e in both lean and DIO models.\u003c/p\u003e\u003cp\u003eEvaluation of myeloid cell infiltration in the 2838c3 TME, revealed a significant increase in proportions of Ly6C\u003csup\u003eint\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e gMDSC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and increased expression of PD-L1 and CD206 on gMDSC within 2838c3 DIO tumors after MRTX1133 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). No significant changes were noted in abundances of CD11b\u003csup\u003e+\u003c/sup\u003e Ly6G/C\u003csup\u003e\u0026minus;\u003c/sup\u003e F4/80\u003csup\u003e+\u003c/sup\u003e macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), however, an increase in proportions of MHCII\u003csup\u003e+\u003c/sup\u003e CD206\u003csup\u003e\u0026minus;\u003c/sup\u003e pro-inflammatory M1 like macrophage in lean 2838c3 tumors and a decrease in MHCII\u003csup\u003e\u0026minus;/+\u003c/sup\u003e CD206\u003csup\u003e+\u003c/sup\u003e anti-inflammatory M2 like macrophage was noted with MRTX1133 treatment in both lean and DIO tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In comparison to 2838c3, minimal differences in abundance of gMDSCs (already elevated) and F4/80\u003csup\u003e+\u003c/sup\u003e macrophage were observed in 6419c5 (both lean and DIO) with MRTX1133 treatment (\u003cb\u003eSupplementary Fig.\u0026nbsp;2a-d\u003c/b\u003e). However, similar to 2838c3, MRTX1133 treatment induced M1 macrophage polarization (\u003cb\u003eSupplementary Fig.\u0026nbsp;2e\u003c/b\u003e) implying similar impact of MRTX1133 inhibition of oncogenic Kras\u003csup\u003eG12D\u003c/sup\u003e signaling on macrophage polarization in both models\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo better understand the impact of DIO on innate cell changes in the DIO 2838c3 TME, we performed analysis of differential gene expression in 2838c3 lean and DIO models treated with MRTX1133 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Significant increases in CD36 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-f) and mRNAs ANGPTL4, COL11A1, and SERPINB5, which are involved directly in promoting tumor proliferation, migration, metastasis \u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and EMT \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, were noted. Interestingly, CD36 is expressed on a wider range of cells within tumor microenvironment including tumor, stromal, regulatory T cells, CAFs and immunosuppressive myeloid cells \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and elevated CD36 mRNA in PDAC patients is associated with poor outcomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Previous studies support a role for CD36 in recruitment, metabolic reprogramming, and immunosuppressive activities of gMDSC\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e suggesting that DIO impacts gMDSC recruitment into the 2838c3 TME and may involve CD36 signaling. Overall, these findings suggest potential tumor-, stromal-, and immune-specific adaptations in DIO mice in response to MRTX1133 that have the potential to impact treatment outcomes.\u003c/p\u003e\n\u003ch3\u003eDIO reduces T cell infiltration and cytotoxic effector activity of T cells post MRTX1133 treatment\u003c/h3\u003e\n\u003cp\u003ePrevious work indicates a correlation between increasing gMDSC infiltration into PDAC TME and a reduction CD8\u003csup\u003e+\u003c/sup\u003e T cell abundance and function\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Therefore, we evaluated the impact of DIO on the T cells response to MRTX1133 treatment within the TiME of 2838c3 tumors. Analysis of CD3\u003csup\u003e+\u003c/sup\u003e T cell infiltrate within TiME revealed reductions in CD4\u003csup\u003e+\u003c/sup\u003e Foxp3\u003csup\u003e\u0026minus;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh) and CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei) in MRTX1133-treated, 2838c3 tumors from DIO mice. Furthermore, the ratio of CD8\u003csup\u003e+\u003c/sup\u003e T cells to gMDSC was significantly reduced in MRTX1133-treated 2838c3 tumors from DIO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej) suggesting an important imbalance in cytotoxic T cells to gMDSC abundances. Analysis of T cell activation (PD-1, CD69, CD44, CD62L) and proliferation (Ki67) revealed significant increases in PD-1 (\u003cb\u003eSupplementary Fig.\u0026nbsp;3a\u003c/b\u003e), CD69 (\u003cb\u003eSupplementary Fig.\u0026nbsp;3b\u003c/b\u003e\u003cem\u003e)\u003c/em\u003e, trending increase of CD44\u003csup\u003ehi\u003c/sup\u003eCD62L\u003csup\u003e\u0026minus;\u003c/sup\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;3c\u003c/b\u003e) abundance and increases in Ki67\u003csup\u003e+\u003c/sup\u003e proliferation (\u003cb\u003eSupplementary Fig.\u0026nbsp;3d\u003c/b\u003e) within CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells supporting a shift to effector T cell phenotype. Furthermore, CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cells isolated from MRTX1133-treated, 2838c3 TME from DIO mice produced less IFNγ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek) after \u003cem\u003ein vitro\u003c/em\u003e stimulation. These findings are consistent with hypothesis that increased gMDSC infiltration into the TME impacts T cell infiltration and function in the TiME and most likely turnover\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e resulting in reductions in T-cell mediated control of microscopic tumor growth. Analysis of mRNA revealed significant downregulation of mRNA associated with T cell abundances, trafficking, retention, and Th1 effector activities including CD3d, CD3g, Foxp3, CXCR3, CXCR6, NKG7, LTB, and DPP4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Pathway and gene ontology analysis revealed downregulation of pathways associated with positive regulation of leukocyte activation, migration, and NK cell immunity (\u003cb\u003eSupplementary Fig.\u0026nbsp;3e\u003c/b\u003e). In comparison to 2838c3 tumors, no significant differences were noted in T or NK cell abundance (\u003cb\u003eSupplementary Fig.\u0026nbsp;4a-bB)\u003c/b\u003e, CD8: gMDSC ratios (\u003cb\u003eSupplementary 4c\u003c/b\u003e), T cell expression of PD-1, CD69, Ki67 (\u003cb\u003eSupplementary Fig.\u0026nbsp;4d-f\u003c/b\u003e) and IFNγ (\u003cb\u003eSupplementary Fig.\u0026nbsp;4g\u003c/b\u003e) in MRTX1133 treatment in 6419c5 lean and DIO cohorts.\u003c/p\u003e\u003cp\u003eOverall, these findings indicate a role for obesity driven inflammation in dampening T cell-mediated control of residual microscopic disease after targeted KRAS\u003csup\u003eG12D\u003c/sup\u003e inhibition that may negatively impact efficacy of parenteral administration of MRTX11333 in obese patients, resulting in greater rates of disease recurrence. Additional studies on the molecular machinery involved, in particular CD36 and ANGPTL4, as promoters of metabolic reprogramming of cancer and immune cells, SERPINB5 and COL11A1, as modulators of stromal responses to treatment and tumor progression, and the impact of MDSC depletion and PD-1 ICB in combination with MRTX1133 in obese models of PDAC is warranted.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure of Potential Conflicts of Interest:\u0026nbsp;\u003c/strong\u003eNo potential conflicts of interest were disclosed by the authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions: \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eConception and design:\u003c/u\u003e Lyse A. Norian and Jeremy B. Foote\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eDevelopment of methodology:\u003c/u\u003e Lyse A. Norian and Jeremy B. Foote\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAcquisition of data:\u003c/u\u003e Sujith Sarvesh, Jeremy B. Foote, Cherlene Hardy, Pia Muri, Henry Nnaemeka Ogbonna, Lyse A. Norian\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAnalysis and interpretation of data:\u003c/u\u003e Sujith Sarvesh, Holly Stephens, Jianqing Zhang, Lyse A. Norian and Jeremy B. Foote\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eWriting, review, and/or editing of manuscript:\u003c/u\u003e Sujith Sarvesh, Dhana Sekar Reddy Bandi, Bassel F. El-Rayes, Lyse A. Norian, and Jeremy B. Foote\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAdministrative, technical, or material support:\u003c/u\u003e Bassel F. El-Rayes, Ganji Purnachandra Nagaraju, Lyse A. Norian, and Jeremy B. Foote\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eStudy Supervision:\u003c/u\u003e Lyse A. Norian and Jeremy Foote\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e Research histology services were provided by the Comparative Pathology Laboratory (Animal Resources Program), NanoString by UAB NanoString Laboratory (Department of Radiation Oncology) and Flow Cytometry support by the UAB Flow Cytometry Core (S10 OD032296).\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The research was funded by an intramural 2022-2023 pilot grant award to Lyse A. Norian and Jeremy B. Foote from the UAB Departments of Microbiology and the Nutrition Obesity Research Center (NORC) for the grant proposal application “Targeted Inhibition of Oncogenic KRAS in Lean and Obese Pancreatic Ductal Adenocarcinoma Tumor Immune Microenivornment”. \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eAnimals\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEight-week-old female C57BL/6 mice (000664) mice were purchased from Jackson Laboratory and used for these studies to reduce incidence of aggression and injury seen in male mice when fed high fat diets. All animal experiments and procedures were performed in accordance with guidelines from the University of Alabama at Birmingham\u0026rsquo;s Institutional Animal Care \u0026amp; Use Committee (IACUC) using an approved IACUC protocol (APN22661). \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDiet\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFollowing one week of acclimation, animals were randomized to one of two matched, purified-ingredient diets: a low-fat diet (LFD; Research Diets 12450J with 10% kcal from fat and matching sucrose to 12492) or high-fat diet (HFD; Research Diets 12492 with 60% kcal from fat) for 12-14 weeks, thereby generating lean (LFD) or diet-induced obese (DIO, HFD) mice. Mice were weighed weekly and fasted for 24 hours followed by an assessment of blood glucose levels. Prior to use, individual mice were identified as DIO if their final body weight was \u0026gt; 3 s.d. above the mean of age-matched lean (LFD-fed) mice within the same cohort, as per our reported methods\u003csup\u003e32\u003c/sup\u003e. Before tumor cell implantation and initiation of \u003cem\u003ein vivo \u003c/em\u003estudies, we established mice with the appropriate body conditions by feeding either low fat diet (LFD; 10% kcal from fat) or high fat diet (HFD, 60% kcal from fat) for 12 weeks\u003csup\u003e28\u003c/sup\u003e . After this, mice were weighed and classified as either lean (LFD, final body weight \u0026lt; 25 grams) or DIO (HFD, final body weight \u0026gt; 3 s.d. above the LFD mean) based on established metrics\u003csup\u003e18,28\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCell lines\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMurine PDAC cell lines purchased from Kerafast (Boston, MA) were derived by Li et al.\u003csup\u003e21\u003c/sup\u003e from tumor-bearing Kras\u003csup\u003eLSL-G12D/+\u003c/sup\u003e; Trp53\u003csup\u003eLSL-R172H/+\u003c/sup\u003e; Pdx1-Cre; Rosa26\u003csup\u003eYFP/YFP\u003c/sup\u003e (6419c5, 2838c3) mice and cultured in DMEM complete media as previously indicated\u003csup\u003e29\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTumor cell implantation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA single-cell suspension of murine PDAC cells was prepared in PBS and kept on ice until injection. Tumor cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) were injected subcutaneously into the flank regions of 8-week-old, female C57BL/6 mice.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMRTX1133 formulation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor in vitro treatments, MRTX1133 (Chemitek, Indianapolis IN) was dissolved in DMSO at a concentration of 25 \u0026mu;M and diluted to 10, 100, 200, 500, 1000 \u0026eta;M concentrations in DMEM complete media. PDAC tumor cell lines (2838c3 and 6419c5) were plated at 1.5 x 10\u003csup\u003e5\u003c/sup\u003e cells/well in 6-well plates and incubated in working concentrations of MRTX1133 above for either 3 or 24 hours. Cells were then washed 3x in PBS and lysates were generated using RIPA buffer with protease and phosphatase inhibitors for downstream immunoblotting. \u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e studies MRTX1133 was formulated in 10% research grade Captisol (CyDex Pharmaceuticals) in 50 mmol/L citrate buffer pH 5.0. Once formulated, MRTX1133 was protected from light and stored at 4\u0026deg;C for 1 week treatment course. MRTX1133 was administered at 30 mg/kg via i.p. injection with b.i.d. dosing either daily (tumor growth curves) after tumors reached an average volume of 50-70 mm\u003csup\u003e3\u003c/sup\u003e or in a modified dosing schedule (1 day SID, 3 days BID, 1 day rest, 1 day SID at endpoint) in order to preserve tumor for downstream flow cytometry profiling of the immune microenvironment. After terminating treatment with MRTX1133 at day 8, MRTX1133-treated 2838c3 lean and DIO cohorts were monitored for 60 days using bi-weekly measurements of tumors. Mice with tumor implantation sites containing no palpable evidence of tumor mass (\u0026lt; 3 x 3 mm) were considered to lack gross tumor burden (complete regression). \u003c/p\u003e\n\u003cp\u003ePrior to determining the impact of DIO on MDSC recruitment, we adjusted the dosing schedule to ensure sufficient 2838c3 tumor tissue was present for downstream analysis after termination of MRTX1133 treatment. The following changes to the MRTX1133 treatment regimen were used with YFP-expressing 2838c3 and 6419c5 cells: (i) tumors were grown for a longer period of time (~14 days) to generate larger tumors (~ 300 \u0026ndash; 700 mm\u003csup\u003e3\u003c/sup\u003e) prior to treatment and (ii) the treatment schedule was abbreviated to an initial PM dose on day 1, AM/PM dosing on days 2-4, a day of rest on day 5, followed by an AM dose on day 6, exactly 3 hours prior to tissue collection for FACS, NanoString, and histology.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNanoString and Gene Ontology Analysis of Tumor Immune Microenvironment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAt day 20 experimental endpoint, 2838ce and 6419c5 PDAC tumors were excised and a portion was preserved in RNAlater. RNA was batch isolated and submitted to the UAB NanoString facility for interrogation using the nCounter PanCancer Immune Profiling Panel (XT-CSO-MIP1-12) according to the manufacturer\u0026rsquo;s instructions. Data analysis was performed using the Rosalind Bioinformatics Analysis Platform for NanoString nCounter Gene Analysis. Differentially expressed genes were identified as those with a p value \u0026lt; 0.05 and fold change \u0026gt; \u0026plusmn; 1.5. Due to the exploratory nature of our study and intent for validation, we used raw p values and did not adjust p values for multiple comparisons.\u003c/p\u003e\n\u003cp\u003eThe NanoString tumor immunogenetic profiling dataset expression matrix was composed of n=12 mice bearing 2838c3 tumors consisting of the following groups: (i) vehicle treated, lean body mass, (ii) MRTX1133 treated, lean body mass, (iii) vehicle treated, DIO, and (iv) MRTX1133 treated, DIO mice. Differentially expressed genes were separated into groups of upregulated or downregulated genes and subjected to Gene Ontology enrichment analysis using the PANTHER classification system to identify associated biological processes (http://geneontology.org/). \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmunoblotting\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTumor or cell lysates were generated after treatment with RIPA lysis buffer (Thermoscientific, IL, USA) with protease and phosphatase inhibitors (Milipore, MA, USA) and sonicated for homogenization and equal protein concentrations were loaded and run on a 4-20% SDS-PAGE. Gels were transferred to a PVDF membrane, which were blocked with or 5% BSA in TBS-T (TBS with 0.1% Tween 20) depending on the blocking agent used for the primary antibodies, for 1 hour at room temperature. Membranes were incubated overnight at 4\u0026deg;C on a rocker with primary antibodies (\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e) according to the manufacturer\u0026rsquo;s instructions and then incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology) for 45 min at room temperature on an orbital shaker. Membranes were then washed to remove excess secondary antibody and developed using SuperSignal West Enhanced Chemiluminescent substrate (Thermo Fisher Scientific) and imaged using a Odyssey XF (Li-Cor). Representative whole blots (pERK, tERK, \u0026beta;actin) for 2838c3 (\u003cstrong\u003eSupplementary Figure 5\u003c/strong\u003e) and 6419c5 (\u003cstrong\u003eSupplementary Figure 6\u003c/strong\u003e) at 3 hours post incubation in vehicle/MRTX1133 are included. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMultiparameter Flow Cytometry\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMouse tumors were digested in a 1 mg/mL collagenase IV and 0.1 mg/mL DNAase 1 (Worthington Biochemical, Lakewood, NJ) in HBSS for 45 minutes at 37 \u0026deg;C with intermittent shaking. Samples were then washed with RPMI containing 10% FBS and filtered through a 70\u0026mu;M strainer generating single cell suspensions. Cells were labeled with primary fluorophore-conjugated antibodies and a live/dead stain (\u003cstrong\u003eSupplementary Table 2\u003c/strong\u003e) for 30-60 min at 4 \u0026deg;C, washed and re-suspended in flow buffer(2% Fetal bovine serum in DPBS). Cytokine expression from single cell suspensions of spleen and tumor was quantified as follows: cells were plated at a concentration of 10\u003csup\u003e6\u003c/sup\u003e cells in 1 mL of RP-10(RPMI + 10% FBS) media and stimulated for 5 hours at 37 \u0026deg;C using a 1x solution of a Cell Activation Cocktail containing PMA ionomycin, and Brefeldin A (Biolegend, San Diego, CA). Conversely, cells were cultured for 5 hours at 37\u0026deg;C using a solution containing 5 ug of Brefeldin A (BioLegend, San Diego, CA) to serve as an unstimulated control. After cell surface staining, cells were fixed in 4% paraformaldehyde for 45 minutes at 4 \u0026deg;C then washed with 1x Perm/Wash (BD, 554723). For intracellular staining in tumor and spleen samples, cells were stained with an antibody cocktail specific intracellular cytokines (IL-2, IFN\u0026gamma;, TNF\u0026alpha;, IL-17A, and granzyme B) in Perm/Wash solution for 60 minutes at 4 \u0026deg;C. Data were acquired on a Symphony A5 flow cytometer, with analysis performed using FlowJo version 10.7.2.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical Analyses\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGraphPad Prism (version 9.1.2) was used for statistical analyses and graphical representation with data are presented as either means \u0026plusmn; standard deviation (SD) or standard error of the mean (SEM). Two-tailed Student\u0026rsquo;s t-test and Two-way Analysis of Variance (ANOVA) with multiple corrections (Bonnferoni method) were performed for determination of statistical significance between groups. \u003cem\u003e\u003cbr\u003e \u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Kratzer TB, Giaquinto AN, Sung H, Jemal A. Cancer statistics, 2025. \u003cem\u003eCa\u003c/em\u003e. 2025;75(1):10.\u003c/li\u003e\n\u003cli\u003eKaramitopoulou E. 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Tumor cell-intrinsic factors underlie heterogeneity of immune cell infiltration and response to immunotherapy. \u003cem\u003eImmunity\u003c/em\u003e. 2018;49(1):178-193. e7.\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":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-precision-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjprecisiononcology","sideBox":"Learn more about [npj Precision Oncology](http://www.nature.com/npjprecisiononcology/)","snPcode":"41698","submissionUrl":"https://submission.springernature.com/new-submission/41698/3","title":"npj Precision Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7871709/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7871709/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiet-induced obesity (DIO) promotes pancreatic tumor progression, immunosuppression, and resistance to chemotherapy. The impact of DIO on efficacy of KRAS\u003csup\u003eG12D\u003c/sup\u003e-specific inhibitor MRTX1133 in syngeneic mouse PDAC cell lines implanted into in lean and DIO mice was evaluated. MRTX1133 induced regression in syngeneic murine PDAC models, irrespective of immune microenvironment composition or body condition, yet, DIO promoted immunosuppression through recruitment of gMDSC into T-cell inflamed TME, reversing control of microscopic disease.\u003c/p\u003e","manuscriptTitle":"Variable Efficacy of the Non-covalent KRAS G12D Inhibitor (MRTX-1133) with Obesity in Murine Pancreatic Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-06 09:29:42","doi":"10.21203/rs.3.rs-7871709/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-17T15:28:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-11T23:02:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216664941011525380276605718449551822809","date":"2025-11-01T16:05:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-29T17:00:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-28T20:37:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28782275319754297712573341945301178483","date":"2025-10-27T19:59:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321581326962528517963317940206844675726","date":"2025-10-27T16:44:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132381581821442556210890312300189520352","date":"2025-10-27T16:13:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-27T15:48:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-26T21:00:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-26T15:43:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Precision Oncology","date":"2025-10-15T21:21:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-precision-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjprecisiononcology","sideBox":"Learn more about [npj Precision Oncology](http://www.nature.com/npjprecisiononcology/)","snPcode":"41698","submissionUrl":"https://submission.springernature.com/new-submission/41698/3","title":"npj Precision Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ee174c0e-f805-49ca-8cec-a75a562061a2","owner":[],"postedDate":"November 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57276433,"name":"Biological sciences/Cancer"},{"id":57276434,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-05-11T13:57:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-06 09:29:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7871709","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7871709","identity":"rs-7871709","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}