Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disease caused by mutations in the dystrophin gene, resulting in severe muscle weakness, degeneration, and early death. Abnormal phenotypes seen in DMD muscle include inflammation, fibrosis, and dysregulated metabolism. Literature suggests microRNA-146a (miR-146a) targets each of these phenotypes: inflammation via nuclear factor-kappa B, fibrosis via transforming growth factor-beta, and metabolism via peroxisome proliferator-activated receptor (Ppar)-alpha, Ppar-gamma, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha. We therefore hypothesize that overexpression of miR-146a will reduce muscle inflammation and fibrosis in dystrophic mice and improve muscle metabolic defects, resulting in a stabilized muscle environment. We generated a cohort of dystrophin-deficient (mdx4cv) mice overexpressing miR-146a (mdx4cv;miR-146aKI/WT) and analyzed muscle histology at 3 and 7 months of age compared to mdx4cv mice. Data show reduced myofiber degeneration in skeletal muscle of mdx4cv;miR-146aKI/WT mice at 3 months of age and reduced cumulative muscle damage in the diaphragm muscle of mdx4cv;miR-146aKI/WT mice by 7 months of age. We performed spatial transcriptomics on diaphragm muscles of 7-month-old dystrophic mice and demonstrated that genes responsible for inflammation are downregulated with miR-146a overexpression, whereas driver genes for pathways involved in muscle function and oxidative phosphorylation (metabolism) are upregulated. Collectively, our data show an exciting potential for miR-146a as a therapeutic to protect muscle from deleterious downstream effects of dystrophin-deficiency. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Engineering a KrasLSL-G12R/+ murine model.

The frequency of co-mutations in tumor suppressor genes is not dependent on KRAS mutation status in human pancreatic cancer patients in the AACR Project GENIE dataset.

Abstract Growth Differentiation Factor 15 (GDF15) is a stress-induced cytokine upregulated in several cancers, including pancreatic cancer. Its most well-known role is signaling through GFRAL in the brain to induce an anorexia/cachexia syndrome. Outside the brain, GDF15 acts as an immunosuppressor. In cancer, GDF15 is thought to leverage this function to protect tumors from immune surveillance. Recently, much attention has been drawn to the relationship between GDF15 and T cell activity, as blocking GDF15 has been shown to enhance responses to anti-PD-1 therapies. Interestingly, the only known receptor for GDF15 is GFRAL, whose expression is restricted to a subpopulation of neurons located in the brainstem. As GDF15 emerges as a target for enhancing immune cell activity, the role of GFRAL in this relationship remains underexplored in animal models of cancer. The purpose of this study is to evaluate the necessity of GFRAL in mediating the immunosuppressive activity of GDF15 with relevance to pancreatic ductal adenocarcinoma (PDAC), the most aggressive form of pancreatic cancer. To test this, T cells were isolated from the whole spleen of immunocompetent mice and activated in the presence of conditioned media (CM) from murine KPC PDAC cells (PdxCre; Kras+/G12D; Trp53fl/fl). In culture, we find that CM containing GDF15 suppressed the proliferation of CD4 and CD8 T cells. qRT-PCR confirmed that T cells lack GFRAL expression, suggesting that GDF15-mediated suppression of T cell activity does not require its canonical receptor. Additionally, KPC CM equally inhibited the proliferation of T cells isolated from GFRAL+/+ and GFRAL−/- mice. We previously showed that GDF15 is required for early-stage development of pancreatic cancer in mice. To determine if this function of GDF15 is mediated through GFRAL, KPC cells were orthotopically injected into the pancreas of immunocompetent GFRAL+/+ and GFRAL−/- mice. No significant differences in tumor burden or overall survival were observed between GFRAL+/+ and GFRAL−/- mice at the experimental endpoint. In addition, both GFRAL+/+ and GFRAL−/- mice exhibited similar myeloid and lymphoid immune responses to tumor implantation compared to non-tumor controls. Moreover, expression of the T cell exhaustion marker PD-1 remained consistent between groups. These results suggest that GDF15 modulates the tumor microenvironment independently of its sole known receptor, GFRAL. Further, disruption of the GDF15-GFRAL signaling axis does not alter the immune cell landscape or enhance T cell expansion. These findings provide valuable insights into the immunomodulatory function of GDF15 in PDAC and the possible distinct roles that the GDF15-GFRAL signaling axis has in regulating anorexia/cachexia vs tumorigenesis. Citation Format: Emma Crockett Funk, Abasi-ama Udeme, Cynthia F. Wright, Jenna Schwesig, Michael C. Ostrowski, Teresa A. Zimmers, Leonidas G. Koniaris, David Wang, Denis C. Guttridge. GFRAL is not required for PDAC tumor development and TME immune cell suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4999.

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumor types, claiming approximately 50,000 lives per year in the United States and carrying a 5-year overall survival rate of only 13%. While immunotherapy has revolutionized therapy for certain cancer types, this has not been the case for PDAC, where there have been zero immunotherapy approvals. This is attributed to a variety of factors related to the PDAC tumor microenvironment (TME) including its dense desmoplastic stroma with high interstitial pressure and a prevalence of immunosuppressive cell types. In addition, genetic characteristics like microsatellite instability (MSI) and high mutational burden, which predict responsiveness to immune checkpoint inhibitors (ICIs), are absent or low in PDAC compared to more responsive tumor types. This suggests that effective anti-tumor immune responses depend on tumor-derived immunogenic signals, leading us to explore new strategies to stimulate immunity against PDAC-associated antigens. We developed a Major Histocompatibility Complex class II (MHCII) targeted approach to direct the immune system against PDAC associated antigens. MHCII is a major antigen presentation complex that directly educates CD4+ helper T cells, and studies have shown that MHCII-CD4 signaling is essential for effective anti-cancer immunity. Specifically, we developed a recombinant high affinity MHCII engager that is conjugated to mesothelin (MSLN), which is overexpressed in a high percentage of PDAC but not the normal pancreas. Methods: To evaluate the preclinical efficacy of our MHCII targeted MSLN therapy (LTI-002), we injected KPC mouse PDAC cells orthotopically in syngeneic C57BL/6 mice. Tumor burden was measured at a predetermined endpoint or by survival analysis. Pharmacodynamic markers of immune response including anti-MSLN specific IgGs and antigen-specific T cell recall were also evaluated. Results: This approach induced a polyclonal humoral response and antigen specific T cell response against mouse and human MSLN candidates. This led to a significant reduction in the size of orthotopic PDAC tumors and significantly improved animal survival. Anti-tumor responses were detected in therapeutic as well as prophylactic experiments and further demonstrated in the de novo KPF mouse PDAC model and in a model of PDAC metastasis. We observed synergy in combination with PDAC standard of care agents like gemcitabine, RAS(ON) inhibitors, and anti-PD1 immunotherapy. As a direct MHCII engager, LTI-002 induced internalization of MSLN into early endosomes where it was processed and loaded onto new MHCII molecules for presentation to CD4+ T cells. LTI-002 target engagement was independent of MHCII polymorphisms, which is an advantage given the diversity of HLA alleles found in different human populations. Conclusions: This study demonstrates the therapeutic potential of targeting MHCII to stimulate anti-PDAC immunity. Citation Format: Reeder Robinson, Leticia Reyes, Samaneh Saberi, Lena Golick, Sandeep Gupta, Michael Ostrowski, Nathan G. Dolloff. MHC class II targeted immunotherapy in the treatment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5551.

High-fat diet (HFD) and associated obesity are suggested to predispose to cancer development, complicate cancer treatment, and accelerate mortality. Paradoxically, obese patients with lung cancer are reported to live longer, suggesting that high body mass is protective. Given that cachexia-tumor-induced weight loss with adipose and muscle wasting-is prevalent in lung cancer, we speculated that patients with obesity might survive longer due to the protective effect of larger tissue reservoirs, slowing time to fatal wasting. Thus, we modeled this condition using lean and high-fat diet (HFD)-induced obese mice with Lewis lung carcinoma (LLC) tumors versus nontumor-bearing controls. We also assessed the effects of feeding HFD to lean mice with and without LLC tumors. HFD and obese-HFD mice without tumors gained weight over the study, with obese-HFD mice exhibiting low muscle mass with obesity at endpoint. Low-fat diet (LFD)-fed lean mice with LLC tumors (LFD-LLC) showed no change in total body weight, but exhibited reduced skeletal muscle, heart, and fat pad mass along with hepatosplenomegaly at endpoint. HFD and pre-existing obesity both modified the response to Lewis lung carcinoma (LLC) tumors. HFD did not affect tumor-induced weight loss, fat loss, or tumor burden, but worsened loss of gastrocnemius, tibialis anterior, and heart muscle, prevented hepatosplenomegaly, and enhanced tumor cell proliferation and expression of the cachexia-inducing cytokine, interleukin-6 (IL-6). Obese-HFD mice showed greater tumor burden versus LFD and the worst cachexia phenotypes, including greater weight loss and muscle loss than HFD or LFD. This worsened cachexia was associated with increased blood-borne inflammatory cytokines, increased phosphorylated STAT3 in muscle, and increased IL-6 expression in muscle, spleen, and tumor. Obese-HFD was associated with the highest rate of tumor cell proliferation in vivo, and serum from obese HFD mice increased LLC cell proliferation in vitro. Thus, HFD and pre-existing obesity each separately enhance inflammation, cachexia, and tumor growth. These distinct contributions of HFD and chronic adiposity are potential therapeutic targets to slow cachexia and tumor growth in cancer. NEW & NOTEWORTHY High-fat diet and obesity are linked to increased cancer risk, but the impact on cachexia development remains unclear. Using mouse models, this study demonstrates high-fat diet and obesity each exacerbate muscle wasting, tumor growth, and tumor and muscle IL-6 expression. Our study reveals distinct, overlapping effects with implications for cancer and cachexia interception.

Objectives/Goals: Cancer cachexia is a wasting condition common in pancreatic cancer, marked by loss of muscle, fat, and bone and driven by inflammation. Immune signals contribute to this damage. This study examines how muscle-resident macrophages may regulate muscle loss in pancreatic cancer cachexia. Methods/Study Population: To identify resident macrophages in cachectic muscle, Lyve1Cre-Rosa26;TdTomato mice were orthotopically transplanted with KPCML1 pancreatic cancer cells. Resident macrophages were analyzed via flow cytometry and immunohistochemistry. For muscle-specific macrophage ablation, lyve1-DTR mice (C57BL/6 background) received tumor cell injections followed by intramuscular diphtheria toxin (DTA) in the TA muscle. Quantitative RT-PCR assessed gene expression from whole muscle lysates and flow-sorted TdTomato+ cells. Results/Anticipated Results: We show that the population of muscle-resident macrophages increased as tumor burden increased. Further findings reveal that the ablation of the resident population of macrophages exacerbates muscle loss, suggesting that muscle-resident macrophages exude a protective phenotype on skeletal myofibers. This was confirmed with increased expression of atrophy biomarkers, MuRF1 and Atrogin-1, in muscles from DTA-injected mice. Additionally, preliminary data revealed an expansion of Toll-like receptor 7 (TLR7) expression in muscle-resident macrophages, suggesting that TLR7 signaling may contribute to their phenotypic regulation in cachexia. Discussion/Significance of Impact: Results suggest muscle-resident macrophages protect against cachexia-induced atrophy via anti-inflammatory activity. Future work will define their signals and targets. Given TLR7 upregulation, we will test if TLR7 signaling regulates their phenotype and contributes to muscle atrophy.

KRASG12R-mutant cell lines have reduced migration.

KrasLSL-G12D/+;Trp53LSL-R172H/+;p48LSL-Cre-ERTM promotes PDAC and liver metastases whereas KrasLSL-G12R/+;Trp53LSL-R172H/+;p48LSL-Cre-ERTM drives inflammation.

Patients with pancreatic ductal adenocarcinoma (PDAC) harboring KRASG12R mutations have increased overall survival relative to patients with KRASG12D/V mutations. To investigate the mechanisms underlying this differential outcome, we developed a genetically engineered mouse model (GEMM) harboring KrasG12R and Trp53R172H mutations (KrasLSL-G12R/+;Trp53LSL-R172H/+;p48Cre-ERTM). Unlike KrasG12D models, KrasG12R GEMMs exhibited limited tumorigenesis, with only 10% developing pancreatic tumors after 1 year. Additionally, mice harboring whole-body expression of KrasG12R remained healthy for over 1 year, whereas KrasG12D mice developed rapid multifocal disease. Comparison of KRAS mutant-selective transcription and signaling in murine and human PDAC cell lines, GEMMs, and patient-derived xenograft (PDX) mouse models revealed that direct KRAS-mediated PI3K activation is necessary for robust tumor initiation in GEMMs. Unexpectedly, KRAS was not the primary driver of PI3K activity in human PDAC cell lines and PDX models, regardless of KRAS mutation. KRASG12R and KRASG12D activated a similar pancreas-specific transcriptional network, but KRASG12R promoted these pathways less robustly due to limited ERK/MAPK nuclear translocation. Finally, KRASG12R human pancreatic tumors had an altered tumor microenvironment (TME) with reduced collagen deposition and metastatic liver invasion. Together, this study demonstrated that KRASG12R is capable of driving tumorigenesis despite the reduced ERK/MAPK nuclear translocation and transcriptional output. Although human KRASG12D- and KRASG12R-mutant tumors display unexpected similarities in PI3K activity, the differential ERK/MAPK signaling activity and the extrinsic consequences on the TME provide support for using KRASG12R mutation status as a prognostic biomarker for therapeutic strategies. SIGNIFICANCE:KRASG12R-mutant pancreatic cancer is characterized by lower ERK/MAPK nuclear translocation and transcriptional output than KRASG12D-mutant tumors, offering a potential window for patients with KRASG12R mutations to derive additional benefit from neoadjuvant therapy. See related commentary by Tiriac and Engle, p. 1817 See related article by Burge et al., p. 1854 See related article by Kamgar et al., p. 2042.

Skeletal muscle loss in pancreatic cancer is a significant cause of morbidity and mortality for patients. In order to understand myocytes changes we examined myonuclei- and myofiber-specific dynamics during pancreatic cancer cachexia progression. Single-nucleus RNA-seq was used to interrogate myonuclear gene expression, and RNAscope and immunofluorescence characterized myofiber-specific changes. Bulk RNA-seq of skeletal muscle provided a whole-muscle transcriptomic profile. Cachexia induces a progressive loss of muscle differentiation factor Maf and its target Myh4 , accompanied by increased expression of Myh1 and Myh2 . This myofiber dedifferentiation occurs without evidence for fiber type shifting, regeneration, or proliferation. Single-nuclei analysis reveals global shifts in myofiber gene expression identity including the identification of a cachexia only myonuclear subpopulation. Cachexia gene expression was not restricted solely to this PDAC-specific myonuclear subpopulation and did not overlap with Myh1 and Myh2 expressing myonuclei early in cachexia. Altogether, PDAC cachexia elicits distinct transcriptional responses across different myonuclear populations. These results reveal population-specific heterogeneity in cachexia gene activation, rather than a uniform upregulation of cachexia mediators across muscle tissue. Our data suggest that myonuclei fate occurs prior to overt muscle wasting when cachexia gene expression only modestly overlaps with differentiation factors, with a strong association after irreversible muscle wasting. These findings explain the challenge of effectively targeting skeletal muscle wasting in cancer cachexia requires addressing the changing cell population induced through non overlapping mechanisms.

Biliverdin reductase A (BVRA), the terminal enzyme in heme catabolism, generates the neuroprotective and lipophilic antioxidant bilirubin. Here, we identify a nonenzymatic role for BVRA in redox regulation. Through phylogenetic, genetic, biochemical, and enzymatic assays, we found that BVRA exerts critical nonenzymatic antioxidant activity. Transcriptomic analyses further revealed that BVRA physically and genetically interacts with nuclear factor erythroid-derived factor-like 2 (NRF2), a major transcriptional regulator of cellular redox signaling. ChIP-seq and RNA-seq analyses reveal that BVRA and NRF2 coordinate the expression of antioxidant genes, many of which are typically dysregulated in neurodegenerative conditions such as Alzheimer's disease. Thus, this noncanonical BVRA-NRF2 axis controls an essential pathway of redox signaling in neuroprotection. Our findings position BVRA as a dual-function integrator of antioxidant defense across both lipophilic and hydrophilic compartments, bridging these two distinct modes of redox protection in the brain.

Lithogenic diet exposure disrupts biliary lipid homeostasis to promote precipitation of excess biliary cholesterol; however, the underlying pathogenic signaling mechanism remains unclear. Protein kinase Cbeta (PKCβ) is involved in regulating hepatic cholesterol and bile acid metabolism. In this study, we aimed to identify the initiating signaling and biological changes in the liver upon loss of hepatic PKCβ function under lithogenic stress. Transcriptome analysis of the liver revealed that hepatic deletion of PKCβ altered the expression of 183 liver genes, 118 of which were upregulated and 65 were downregulated. We identified marked increases in the expression of genes involved in bile acid biosynthesis (Cyp7a1 and Cyp8b1) and a decrease in retinol metabolism (Cyp26b1) as the most relevant changes, with blunted expression of genes involved in bile acid and phosphatidylcholine transporters. Mechanistic studies revealed that the hepatic PKCβ deficiency was associated with reduced ERK1/2 phosphorylation in concert with increased p38MAPK phosphorylation in the liver. Overexpression of PKCβ in the liver blocked p38MAPK activation as well as resulted in increased ERK1/2 phosphorylation and was accompanied by suppression of both Cyp7a1 and Cyp8b1 expression, demonstrating that hepatic PKCβ functions as a positive regulator of ERK1/2 to suppress the expression of both genes by antagonizing p38MAPK. Furthermore, depletion of liver p38MAPK in PKCβLiv-/- mice resulted in enhanced ERK1/2 phosphorylation and suppression of Cyp7a1 and Cyp8b1 expression. The findings yielded by this study support our understanding of the intricate interplay among PKCβ, p38MAPK, and ERK1/2 signaling in vivo and provide valuable insights into potential therapeutic targets for the development of novel strategies to combat cholelithiasis.NEW & NOTEWORTHY This study underscores the pivotal role of hepatic PKCβ in controlling biliary lipid composition under lithogenic stress. Our findings on the distinct and combined effects of downstream p38MAPK and ERK1/2 offer key insights into the mechanisms driving lithogenic diet-induced dysregulation of biliary lipid composition. This research reveals that the potential of PKCβ/p38MAPK/ERK1/2 signaling axis offers the possibility for the integration of different inputs to modulate the signaling output balancing in a way most appropriate for context.

We hypothesize that SFRP2 is a promising target for Triple Negative Breast Cancer (TNBC). 1. Multiplex immunohistochemistry (IHC) was performed on human TNBC to identify SFRP2 localization in the tumor microenvironment. 2. Tumor associated macrophages (TAMs) were isolated from E0771.LMB breast tumors. TAMs were treated with hSFRP2 mAb (10 µM) or control (10 µM) for 1 h and analyzed by western blot and qRT-PCR for IFN-ϒ. 3 SFRP2 and IFN-ϒ mRNA expression levels were analyzed from the Cancer Genome Atlas (tCGA) for breast cancer patients using least squares-linear regression analysis. 4. PY8119 or E0771.LMB TNBC cells were injected i.v. into mice, and mice were treated with either IgG1 or hSFRP2 mAb every 3 days. Lung metastases were counted after 4 weeks and analyzed by IHC for M1/M2 ratio. 5. MDA-MB-231 TNBC cells were injected into the mammary fat pad, and when tumors were established, mice were treated with IGg1 or hSFRP2 mAb every 3 days i.v. for 79 days and tumor volumes were compared. 6. Wild-type (WT) MDA-MB-231 and doxorubicin-resistant MDA-MB-231 cells were treated with hSFRP2 mAb and apoptosis was compared. 1) Multiplex IHC on human breast tumors showed that SFRP2 localized to tumor cells (87

PURPOSE:This randomized phase-II trial (ClinicalTrials.gov identifier: NCT02767557) compared efficacy of gemcitabine/nab-paclitaxel (Gem/Nab) with or without the anti-interleukin-6 (IL-6) receptor antibody tocilizumab (Toc) for advanced pancreatic cancer (PC). METHODS:A safety cohort received Gem 1,000 mg/m2 and Nab 125 mg/m2 on days 1, 8, and 15, and Toc 8 mg/kg on day 1 for each 28-day cycle. Participants with modified Glasgow prognostic scores of 1 or 2 were randomly assigned 1:1 to receive Gem/Nab/Toc or Gem/Nab. The primary end point was the overall survival (OS) rate at 6 months (OS6). Secondary end points were progression-free survival (PFS), overall response rate (ORR), and safety. Exploratory end points were cachexia, quality of life, and biomarkers, including the cachexia-promoting protein, growth differentiation factor 15 (GDF15). RESULTS:Overall, 147 patients were treated, including six safety cohort participants. The median follow-up period was 8.1 months (IQR, 4.2-13.9). OS6 was 68.6% (95% CI, 56.3 to 78.1) for the Gem/Nab/Toc group and 62.0% (49.6-72.1) for the Gem/Nab group (P = .409). OS for Gem/Nab/Toc versus Gem/Nab improved at 18 months (27.1% v 7.0%, P = .001). No differences in median OS, PFS, or ORR were observed. Incidence of grade-3+ treatment-related adverse events (TrAEs) was 88.1% for Gem/Nab/Toc and 63.4% for Gem/Nab (P < .001). Gem/Nab/Toc decreased muscle loss versus Gem/Nab, with median change +0.1013% versus -3.430% (P = .0012) at 2 months and +0.7044 versus -3.353% (P = .036) at 4 months. Incidence of muscle loss was 43.48% on Gem/Nab/Toc versus 73.52% on Gem/Nab at 2 months (P = .0045) and 41.82% versus 68.75% (P = .0062) at 4 months. GDF15 was not changed by Gem/Nab or Gem/Nab/Toc. CONCLUSION:Although the primary end point was not met and TrAEs were increased by Toc, increased survival at 18 months and reduced muscle wasting support an anticachexia effect of IL-6 blockade independent of GDF15. Further studies could leverage these findings for precision anticachexia therapy.

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly malignancy. Cancer associated fibroblasts (CAFs) play a complex role in the PDAC tumor microenvironment (TME), with a subset contributing significantly to tumor progression. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and its loss is associated with increased tumor aggressiveness. Our group previously demonstrated PTEN loss in SMA+ positive CAFS correlated with worsened outcomes in patients and in mouse PDAC models. Our group previously demonstrated the loss of PTEN in CAFS led to activation of Stat3, resulting in an immunosuppressive microenvironment (Lefler et al, 2022 PMID: 35803738). However, little is known of the signals produced by tumor cells or other cell types in the TME that trigger loss of PTEN in CAFs. This study aims to to elucidate the molecular mechanisms underlying PTEN loss in CAFs in PDAC. To address this gap, we employed single cell RNA sequencing of tumors from a mouse PDAC model and identified a (PDRGFRa+Lys6+Stat3+Pten-low) CAF subpopulation. Comparing normal pancreas epithelial cells to tumor cells in this data set, we identified the IL6 family of cytokines, (IL6; Oncostatin-M, OSM; and Leukemia Inhibitory Factor, LIF) and Transforming Growth Factor beta 1 (TGFb1) as potential ligands that regulated PTEN, while myeloid cells were a source of the IL6 family member Oncostatin M (OSM). Treatment of isolated CAFs with these factors revealed no significant difference in PTEN mRNA expression. Thus, we developed an in vitro reporter system in which a PTEN-GFP fusion protein is expressed in CAFs. Treating these CAFs with IL6, OSM and TGFb1 (but not LIF) led to a significant decrease in GFP fluorescence. Analysis of endogenous PTEN by western blot confirmed these results. We posited that expression of PTEN E3 ligases might be increased by treatment with these factors, leading to destruction of PTEN. In support of this hypothesis, the expression of E3 ligases WWP1 and WWP2 was increased by treatment with IL6 and OSM. Additionally, preliminary data has revealed that PTEN is oxidized in isolated CAFs, which causes loss of PTEN activity and potentially increased protein turnover. Current experiments are aimed at testing these potential mechanisms. To conclude, PTEN is dysregulated post-transcriptionally in CAFs. The mechanism of PTEN dysregulation could be via increased expression of E3 ligases that target PTEN and/or PTEN oxidation. Future efforts will be aimed at testing whether inhibition of these pathways can restore PTEN expression in mouse PDAC models. These pathways and interactions could be further exploited for therapeutic benefit in curbing PDAC progression. Citation Format: Ivo N. Woogeng, Lu Han, Samaneh Saberi, Cameron Bumbleburg, Joseph Beaudet, Sudarshana Sharma, Michael Ostrowski. Dysregulation of PTEN expression in a subset of Cancer Associated Fibroblasts by Tumor Secreted Factors in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A060.

Abstract Mutational activation of the KRAS proto-oncogene is the initiating mutational event in pancreatic ductal adenocarcinoma (PDAC) and ~90% of patients harbor KRAS mutations. PDAC is marred by a 12% five-year overall survival rate, and developing effective therapeutic strategies remains a priority. Accumulating evidence suggests that all KRAS mutations are not created equal. While the KRASG12R mutation is rare in lung and colorectal cancers (<1%), it is the third most common KRAS mutation in PDAC, accounting for approximately 20% of all cases. However, KRASG12R cannot interact with the lipid kinase PI3Ka, a well-characterized RAS effector necessary for KRAS-driven tumorigenesis. Previous studies in mice models of cancer have demonstrated that ablation of the KRAS:PI3Ka interaction limits tumorigenesis. Despite the inability of KRASG12R to activate PI3Ka directly, AKT signaling is robustly activated in KRASG12R-mutant PDAC. The mechanisms that allow the KRASG12R mutant to overcome the inability to activate PI3K and promote PDAC are unclear. We recently developed a Ptf1a-CREERT2;KrasLSL-G12R genetically engineered mouse model to study KRASG12R in an in vivo context. This model does not develop pancreas lesions or tumors. We have uncovered two unique characteristics of human PDAC that we hypothesize allows for the KRASG12R mutation to produce tumors only in human pancreatic tissue. First, we have found that all four PI3K isoforms are overexpressed in human PDAC, and the PI3Kd and PI3Kg isoforms are specifically upregulated in KRASG12R-mutant PDAC. Second, PTEN is oxidized in PDAC. PTEN oxidation results in an intramolecular disulfide bond, which inhibits the phosphatase activity of PTEN and leads to hyperactivated PI3K signaling. Critically, mouse pancreas tissue only expresses the PI3Ka/b isoforms and PTEN is in the reduced state, demonstrating two significant differences between mouse models and human disease. Additionally, we have determined that PTEN becomes fully oxidized in nutrient-restricted medium (low glucose/glutamine), a common strategy employed to mimic the pancreatic tumor microenvironment in cell culture. Using PTEN oxidation-resistant variants, we demonstrate that KRASG12R-mutant PDAC cell lines are reliant on oxidized (inactivated) PTEN for proliferation. To confirm that PTEN inactivation can aid KRASG12R-mediated tumorigenesis in mouse models, we generated at KrasG12R/+;Ptenfl/fl genetically engineered mouse model, and the results of this model will be presented herein. Thus, our data demonstrate that increased PI3K isoform expression, coupled with PTEN oxidation, creates a unique environment that allows KRASG12R to initiate and promote pancreatic tumorigenesis. As PTEN oxidation overcomes the need for KRAS to activate PI3K signaling in PDAC, these results indicate that directly targeting KRAS alone will be insufficient at reducing tumor growth in humans and clinically successful therapeutic strategies will have to develop alternative methods to reduce PI3K signaling in addition to directly targeting KRAS activity. Citation Format: Kamala Sundararaj, Rachel Burge, Samaneh Saberikashani, Lucas Bialousow, Amanda Linke, Merissa Smith, Michael C. Ostrowski, John P. O'Bryan, G. Aaron Hobbs. KRAS mutant-specific interactions reveal mechanisms in pancreatic cancer tumorigenesis and metabolic function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr PR11.

Although cancer cachexia is classically characterized as a systemic inflammatory disorder, emerging evidence indicates that weight loss also associates with local tissue inflammation. We queried the regulation of this inflammation and its causality to cachexia by exploring skeletal muscle, whose atrophy strongly associates with poor outcomes. Using multiple mouse models and patient samples, we show that cachectic muscle is marked by enhanced innate immunity. Nuclear factor κB (NF-κB) activity in multiple cells, including satellite cells, myofibers, and fibro-adipogenic progenitors, promotes macrophage expansion equally derived from infiltrating monocytes and resident cells. Moreover, NF-κB-activated cells and macrophages undergo crosstalk; NF-κB+ cells recruit macrophages to inhibit regeneration and promote atrophy but, interestingly, also protect myofibers, while macrophages stimulate NF-κB+ cells to sustain an inflammatory feedforward loop. Together, we propose that NF-κB functions in multiple cells in the muscle microenvironment to stimulate macrophages that both promote and protect against muscle wasting in cancer.

Abstract The pancreas is composed of the epithelial and mesenchymal cells. While mesenchymal fibroblasts are a minor component of the normal pancreas, fibroblast population expands drastically during tumorigenesis. In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. This study sought to define the origin, heterogeneity and function of pancreatic cancer associated fibroblasts. Recently we performed a series of lineage tracing studies in genetically engineered mouse models. This identified the splanchnic mesenchyme (a tissue layer adjacent to the developing fetal pancreatic epithelium) as the fetal origin of the adult pancreatic resident fibroblasts and pancreatic CAFs. Single cell transcriptomic analysis indicated persistent and dynamic gene expressions along the pancreatic mesenchymal trajectory during development, homeostasis, precancer and cancer. Intriguingly, we found that two splanchnic transcription factors, GATA6 and FOXF1, are expressed in only subsets of adult pancreatic fibroblasts in temporally and spatially distinct patterns. Similar patterns were observed in both mouse models and human patient samples. To determine the role of GATA6 in fibroblasts during tumorigenesis, we constructed a dual DNA recombinase mouse genetic model. DNA recombinase FlpO directs expression of an oncogene Kras (G12D mutation) and loss of a tumor suppressor p53 in pancreatic epithelial cells to induce spontaneous tumor formation in the pancreas, and DNA recombinase Cre deletes Gata6 specifically in fibroblasts. This fibroblast specific Gata6 deletion resulted in altered number and gene expression of CAFs as well as increased tumor burden in the pancreas. This suggests a non-cell autonomous function of GATA6 within CAFs to restrain pancreatic cancer progression. In summary, this study delineated a continuous cell trajectory of the mesenchymal lineage in the pancreas across different life stages. Additionally, this study provides evidence that persistent and selective gene expressions along the mesenchymal trajectory contributes to pancreatic CAF heterogeneity and such persistence may constitute an inherent host defense mechanism to restrain pancreatic cancer. The enhancement of this mechanism could be explored further for therapeutic benefits. Citation Format: Lu Han, Tom Walter, Joseph Beaudet, Caroline Everett, Gustavo Leone, Michael Ostrowski. Persistence of fetal splanchnic gene signature defines a tumor-restraining fibroblast subtype in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A044.