KRASG12R-Mutant Pancreatic Cancer Features Limited ERK/MAPK Transcriptional Activity and a Distinctive Tumor Microenvironment

Cancer Research · 2026-01-13 · 2 citations

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.

De novo design of protein binders that target DELE1 to inhibit the mitochondrial stress response

bioRxiv (Cold Spring Harbor Laboratory) · 2025-12-23

Mitochondrial stress activates the integrated stress response (ISR) through the mitochondrial protein DELE1, which relays stress signals to the cytosolic kinase HRI to induce ATF4. Dysregulation of DELE1-mediated signaling has been implicated in pathological conditions, yet molecular strategies to modulate DELE1 remain unavailable. Here, we report de novo designed proteins that bind DELE1, block its oligomerization, and inhibit DELE1-mediated ISR activation. Several designs form stable complexes with DELE1 and disrupt its oligomerization in vitro while preserving DELE1's ability to bind HRI. In cells, these designs suppress ATF4 induction during mitochondrial stress and impair the recovery of elongated mitochondrial morphology following transient insult. Crystal structure of a representative binder, together with structural modeling and targeted mutagenesis, confirm that the designed proteins engage a critical interface required for DELE1 oligomerization. These findings establish DELE1 as a druggable target and demonstrate that de novo designed proteins offer precise tools to modulate this pathway, providing a foundation for future therapeutic exploration. SIGNIFICANCE: Mitochondrial stress activates the integrated stress response through the signaling protein DELE1, but no molecular tools have been available to directly target DELE1 and selectively modulate this pathway. We developed de novo designed protein binders that recognize a critical oligomerization interface in DELE1, disrupt its assembly, and suppress mitochondrial stress-induced ISR activation in cells. These binders also impair recovery of mitochondrial network morphology following transient stress, linking DELE1 assembly to adaptive remodeling. Our study establishes DELE1 as a tractable and druggable target in mitochondrial stress signaling and demonstrates that de novo protein design can generate precise modulators of intracellular stress-response pathways.

Virginia Journal of Medicine Fall 2025 Medical Student Symposium Special Edition

Libra · 2025-12-21

The Medical Student Summer Research Symposium Special Abstracts Edition of the Virginia Journal of Medicine highlights the scholarly work of medical students who participated in structured summer research programs at the University of Virginia. This collection represents a wide range of inquiry, including basic science, clinical research, medical education, health systems, and community-engaged scholarship, made possible through close faculty mentorship and institutional support. This special edition reflects the mission of student-driven scholarship: to celebrate early academic inquiry, foster collaboration, and provide a platform for emerging physician-scientists to share their work with the broader medical community. Together, these abstracts showcase the curiosity, rigor, and innovation that define the next generation of physicians.

NRF2 regulates lipid droplet dynamics to prevent lipotoxicity

iScience · 2025-06-18 · 5 citations

Lipid droplets (LDs) are dynamic organelles comprising a neutral lipid core encapsulated by a phospholipid monolayer. LD structure and function are influenced by a variety of intrinsic and extrinsic signals, and cells alter LD content and distribution to adapt to their environment. Here, we show that LD content increases in response to stabilization of the transcription factor NRF2 under conditions of lipotoxic stress. Notably, NRF2 activity leads to increased expression of the G0S2, a protein that inhibits ATGL, the enzyme responsible for degradation of triacylglycerol and the release of fatty acids from LDs. Importantly, stabilization of NRF2 in the absence of stress is sufficient to increase LD content, and inhibition of ATGL partially rescues the impact of NRF2 deletion on stress-induced ferroptosis. These data support a model in which stress-induced NRF2 stabilization protects cells against lipotoxicity in part through the sequestration of fatty acids in lipid droplets.

Spatial regulation of mitochondrial membrane potential by α5β1 integrin engagement in collective cell migration

Journal of Cell Science · 2025-04-14 · 2 citations

The mechanistic links between mechanical forces and bioenergetics remain elusive. We report an increase in mitochondrial membrane potential (MMP) along the leading row of collectively migrating Xenopus laevis mesendoderm cells at sites where fibronectin-α5β1 integrin substrate traction stresses are greatest. Real-time metabolic analyses reveal α5β1 integrin-dependent increases in respiration efficiency in cells on fibronectin substrates. Elevation of metabolic activity is reduced following pharmacologic inhibition of focal adhesion kinase (FAK; also known as PTK2) signaling. Attachment of mesendoderm cells to fibronectin fragments that support differing α5β1 integrin conformational and ligand-binding affinity states, increases MMP when both the Arg-Gly-Asp (RGD) and Pro-Pro-Ser-Arg-Asn (PPSRN) synergy sites of fibronectin are engaged by the receptor. Cell stretch on deformable fibronectin substrates also results in a FAK-dependent increase in MMP. Inhibition of MMP or ATP-synthase activity slows collective cell migration velocity in vivo, further suggesting that integrin-dependent adhesion and signaling contribute to metabolic changes. These data highlight an underexplored link between extracellular matrix (ECM)-integrin adhesion and metabolic activity in embryonic cell migration. We propose that fibronectin-integrin adhesion and signaling help shape the metabolic landscape of collectively migrating cells.

Investigating the role of DRP1 in mediating an immune suppressive TME in KRAS mutant pancreatic cancer 2696

The Journal of Immunology · 2025-11-01

Abstract Description Pancreatic cancer is a devastating disease with a dismal 5-year relative survival rate of 13%. Innovative therapies that leverage the immune system to kill cancer hold great promise for treatment. However, pancreatic tumors exhibit dysregulated mitochondrial dynamics, which leads to enhanced cell growth and the development of an immune-suppressive tumor microenvironment (TME), limiting treatment efficacy. We previously demonstrated that mutated Kras, the most common mutation in pancreatic cancer, led to increased activation of DRP1, leading to increased mitochondrial fission, elevated glycolysis, and greater lactate secretion. Yet, the full impact of DRP1 expression on the pancreatic TME remains unclear. We hypothesize that activation of the DRP1 pathway is sufficient to generate an immune-suppressive TME. Knocking out DRP1 in patient-derived cell lines revealed an altered cytokine secretion profile, reduced lactate secretion, and slower tumor growth in vitro and in vivo. DRP1 KO cells exhibited increased IL-6 secretion, and immunohistochemical staining of DRP1 KO tumors demonstrated increased T cell infiltration compared with DRP1-expressing controls. Implications for suppressive fibroblast and myeloid cell recruitment and differentiation will be discussed. A greater understanding of the role of DRP1 in pancreatic cancer may lead to insights regarding oncogene-driven metabolic changes in tumor cells and opportunities for therapeutic targeting. Funding Sources T32CA009109-48 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

PTEN Oxidation Promotes Constitutive PI3K Signaling and Inducible Macropinocytosis in Pancreatic Cancer

Cancer Research · 2025-10-24 · 3 citations

Pancreatic ductal adenocarcinoma (PDAC) is defined by the near universal occurrence of KRAS mutations. The KRASG12R mutation is detected in approximately 15% to 20% of patients with PDAC and rare in other KRAS-mutant cancers. KRASG12R is unable to activate the lipid kinase PIK3CA, suggesting that alternative mechanisms might be employed to activate KRASG12R-independent PI3K signaling in PDAC. In this study, we detected elevated expression of all four PI3K isoforms in PDAC cell lines, with the PIK3CG isoform showing higher overall expression in KRASG12R-mutant PDAC. All four PI3K isoforms contributed to global PI3K signaling, and inhibition of any single isoform was insufficient to limit PDAC proliferation. The combined inhibition of all PI3K isoforms was required to limit proliferation, providing a potential explanation for the limited efficacy of PI3K inhibitors in the clinic. Additionally, PTEN, a negative regulator of PI3K signaling, was inactivated in PDAC by the formation of an intramolecular disulfide, which elevated overall PI3K signaling and reduced the dependency of PI3K signaling on KRAS. Oxidation of PTEN was independent of KRAS mutation status. Finally, nutrient-limiting conditions mimicking the PDAC tumor microenvironment further elevated PTEN oxidation and significantly increased macropinocytosis. Thus, this study uncovered a mechanism that supports elevated PI3K signaling in PDAC, thereby reducing the need for KRAS to directly activate the PI3K pathway. SIGNIFICANCE: PTEN inactivation by intramolecular disulfide bond formation and elevated expression of PI3K isoforms in pancreatic cancer leads to unchecked KRAS-independent PI3K signaling, highlighting the need for therapeutic approaches targeting constitutive PI3K signaling. See related commentary by Tiriac and Engle, p. 1817 See related article by Burge et al., p. 1868 See related article by Kamgar et al., p. 2042.

Abstract LB283: Reactivation of Drp1S616 phosphorylation by c-MYC-CDK4/6 signaling axis plays a functional role in resistance to MEK inhibition in pancreatic cancer cells

Cancer Research · 2025-04-25 · 1 citations

Abstract Mitochondria are highly dynamic organelles that can exhibit a range of phenotypes from highly fragmented to highly networked. Many cancer cells exhibit fragmented mitochondrial networks, and this phenotype supports metabolic reprogramming to promote tumor growth. We previously demonstrated that in Ras mutant tumors, ERK mediated phosphorylation of the mitochondrial fission GTPase Drp1 at S616 leads to an increase in mitochondrial fission activity that is responsible for the fragmented phenotype. We and others have demonstrated that Drp1-mediated mitochondrial fission plays a tumor-promoting role in pancreatic and other Ras-driven cancers, but its role in therapeutic resistance is unknown. The MAPK pathway is the most critical effector pathway downstream of mutant Ras. Despite this, targeting this pathway directly in Ras-driven pancreatic cancer has been unsuccessful due to the development of resistance. In the present study we have generated a panel of patient-derived pancreatic cancer cell lines resistant to the MEK inhibitor trametinib and analyzed their mitochondrial phenotypes. We find that trametinib-resistant pancreatic cancer cells exhibit increased expression and phosphorylation of Drp1 compared to sensitive counterparts, even in the presence of trametinib. Furthermore, quantitative analysis of mitochondrial structure reveals that trametinib-induced mitochondrial restructuring is inhibited in resistant cells. Intriguingly, we have identified CDK4/6 as novel kinase of Drp1. Inhibition of CDK4/6 activity is sufficient to block Drp1 phosphorylation in resistant cells, suggesting that activation of these kinases is driving the reactivation of mitochondrial fission in the absence of MAPK signaling. Importantly, CRISPR-mediated deletion of Drp1 from resistant cells either partially re-sensitizes the cells to trametinib (1 line) or leads to growth inhibition independent of drug treatment (3 lines). These data highlight the importance of Drp1 reactivation in maintaining the growth of trametinib resistant tumor cells. Furthermore, we have identified c-MYC oncoprotein is reactivated in trametinib resistant pancreatic cancer cells and mediates Drp1S616 phosphorylation through CDK4/6 kinases. Together, these findings point to the importance of mitochondrial structure in drug resistance and suggest that inhibition of mitochondrial fission might be a promising therapeutic strategy to combat resistance to MAPK and RAS inhibitors. Citation Format: Salma Sharmin, Jennifer A. Kashatus, Sara J Adair, Todd W Bauer, David F Kashatus. Reactivation of Drp1S616 phosphorylation by c-MYC-CDK4/6 signaling axis plays a functional role in resistance to MEK inhibition in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB283.

Proteome-wide copy-number estimation from transcriptomics

Molecular Systems Biology · 2024-09-27 · 4 citations

Protein copy numbers constrain systems-level properties of regulatory networks, but proportional proteomic data remain scarce compared to RNA-seq. We related mRNA to protein statistically using best-available data from quantitative proteomics and transcriptomics for 4366 genes in 369 cell lines. The approach starts with a protein's median copy number and hierarchically appends mRNA-protein and mRNA-mRNA dependencies to define an optimal gene-specific model linking mRNAs to protein. For dozens of cell lines and primary samples, these protein inferences from mRNA outmatch stringent null models, a count-based protein-abundance repository, empirical mRNA-to-protein ratios, and a proteogenomic DREAM challenge winner. The optimal mRNA-to-protein relationships capture biological processes along with hundreds of known protein-protein complexes, suggesting mechanistic relationships. We use the method to identify a viral-receptor abundance threshold for coxsackievirus B3 susceptibility from 1489 systems-biology infection models parameterized by protein inference. When applied to 796 RNA-seq profiles of breast cancer, inferred copy-number estimates collectively re-classify 26-29% of luminal tumors. By adopting a gene-centered perspective of mRNA-protein covariation across different biological contexts, we achieve accuracies comparable to the technical reproducibility of contemporary proteomics.

HSP70-mediated mitochondrial dynamics and autophagy represent a novel vulnerability in pancreatic cancer

Cell Death and Differentiation · 2024-05-28 · 41 citations

Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is one of the deadliest forms of cancer with limited therapy options. Overexpression of the heat shock protein 70 (HSP70) is a hallmark of cancer that is strongly associated with aggressive disease and worse clinical outcomes. However, the underlying mechanisms by which HSP70 allows tumor cells to thrive under conditions of continuous stress have not been fully described. Here, we report that PDAC has the highest expression of HSP70 relative to normal tissue across all cancers analyzed. Furthermore, HSP70 expression is associated with tumor grade and is further enhanced in metastatic PDAC. We show that genetic or therapeutic ablation of HSP70 alters mitochondrial subcellular localization, impairs mitochondrial dynamics, and promotes mitochondrial swelling to induce apoptosis. Mechanistically, we find that targeting HSP70 suppresses the PTEN-induced kinase 1 (PINK1) mediated phosphorylation of dynamin-related protein 1 (DRP1). Treatment with the HSP70 inhibitor AP-4-139B was efficacious as a single agent in primary and metastatic mouse models of PDAC. In addition, we demonstrate that HSP70 inhibition promotes the AMP-activated protein kinase (AMPK) mediated phosphorylation of Beclin-1, a key regulator of autophagic flux. Accordingly, we find that the autophagy inhibitor hydroxychloroquine (HCQ) enhances the ability of AP-4-139B to mediate anti-tumor activity in vivo. Collectively, our results suggest that HSP70 is a multi-functional driver of tumorigenesis that orchestrates mitochondrial dynamics and autophagy. Moreover, these findings support the rationale for concurrent inhibition of HSP70 and autophagy as a novel therapeutic approach for HSP70-driven PDAC.