Epigenetic changes are a major driver of cancer progression, placing considerable focus on epigenetic regulators as therapeutic targets. Protein arginine methyltransferase 5 (PRMT5) is one such regulator, and numerous PRMT5 inhibitors (PRMT5i) in clinical trials. Despite this, the mechanisms and consequences of PRMT5i-resistance are unknown. Here, we demonstrate that aggressive cancer progression is an inbuilt feature of PRMT5i-resistance acquisition in lung adenocarcinoma (LUAD). Independently-generated resistant cell lines gain dedifferentiation signatures that typify late-stage disease and show increased metastatic potential in vivo. We establish that these state shifts are a direct consequence of PRMT5i action; treatment induces rapid and widespread chromatin rewiring, enabling derepression of late-stage disease states that are stably established in resistant cells. Notably, treatment of lung tumor-bearing mice drives rapid disease advancement without decreasing tumor burden, showing that drug-induced disease progression supersedes any benefits from PRMT5 inhibition in vivo. Furthermore, analyses of human cell lines and patient cohorts supports the notion of PRMT5 inhibition-mediated dedifferentiation. Collectively our data show that PRMT5i can actively promote self-resistance and disease progression in different tumor types. This raises serious concerns for the use of PRMT5i in patients, arguing that clinical studies should consider the possibility of drug-induced plasticity, resistance, and disease advancement.

Abstract Introduction Recent clinical trials have shown that messenger RNA vaccination against neoantigens can activate long-lasting CD8+ T cells against solid tumors. However, frequent doses appear to be needed to maintain this response. It is uncertain whether vaccination alone reliably recruits effector cells into tumors, particularly when antigen expression is low. We hypothesize that mRNA vaccination primes anti-tumor CD8+ cells, but this alone is not sufficient to recruit and sustain these cells within tumors. Methods We used a murine lung adenocarcinoma model; intratracheal lentiviral Cre recombinase was used to initiate KrasG12D/+; Trp53-/- tumors expressing two neoantigens. Mice were dosed intramuscularly with neoantigen-encoding mRNA. We analyzed CD8+ cell trafficking via flow cytometry, in vivo cytotoxicity assays, ex vivo transwell assays, and immunohistochemistry. Intravenous fluorescent labeling was used to track CD8+ T cell recruitment into tumors. Results In tumor-bearing mice, we observed a strong presence of neoantigen-specific CD8+ cells in the spleen one week post-vaccination. The majority of these cells were CX3CR1+ short-lived effectors. This was not seen among other antigen-experienced CD8+ cells in the spleen. However, these vaccine-primed effector cells mainly localized to the lung vasculature with limited tissue infiltration. In naïve mice, 99% of transferred neoantigen-pulsed splenocytes were cleared from the spleen within 20 hours. Conclusion Our data suggest that mRNA vaccination activates a strong effector population capable of clearing targets presenting neoantigens. However, these cells are ineffectively recruited from the vasculature, a potential barrier to strong anti-tumor immunity. We believe this is either due to a natural progression into short-lived effector cells after priming or inadequate signaling for tissue entry. Our current work aims to explore whether introducing a pro-migratory chemokine axis in the lung may improve recruitment of vaccine-primed cells into tumors. Funding Source NIH T32 Training Grant Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Introduction Cancer mutations can give rise to novel tumor-specific peptides (“neoantigens”) that are recognized by T cells to attack tumors. CD4+ T cells cooperate with B cells of related antigen specificity to promote antibody production and enhance antitumor immunity. Paradoxically, tumors are frequently coated with matured antibodies that recognize unmutated epitopes despite the existence of tolerance mechanisms. We hypothesized that neoantigen-specific CD4+ T cells play a key role in coordinating polyfunctional tumor-associated B cell responses. Methods To test this, we utilized an autochthonous KP (KrasG12D/+; tp53-/-) mouse model of lung cancer initiated via delivery of Cre, and leveraged flow cytometric and histological analyses with temporal perturbations, depletions, and ELISA assay. Results Tumors expressing a single MHC-II-restricted neoantigen (mITGB1) or lacking this neoantigen (mITGB1-neg) harbored similar densities of B cells. However, B cells in mITGB1+ tumors uniquely displayed hallmarks of germinal center entry, class switching, and differentiation, and many B cells were spatially confined to clusters containing T cells. B cells persisted in mITGB1+ tumors for at least three weeks when lymphocyte entry was blocked, while B cells in mITGB1-neg tumors disappeared in this setting. Serum IgG isolated from mITGB1+ but not mITGB1-neg tumor-bearing mice showed reactivity against a non-autologous, neoantigen-deficient KP-derived cell line despite the presence of germinal center B cells in the draining lymph nodes of mITGB1-neg tumors. Finally, B cell ablation led to increased tumor burden, loss of intratumoral T cell clusters, and altered T cell composition and positioning in the tumor bed. Conclusion Our data indicate that neoantigen-specific CD4+ T cells orchestrate local B cell responses within tumors to promote anti-tumor immunity. Intratumoral lymphocyte hubs may represent unique sites of B cell selection, and function as a source of cancer-targeting antibodies and antigenic spread. Funding Source Jane Coffin Childs Fund, Hope Funds for Cancer Research Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Age-related blood cell mutations (clonal hematopoiesis) reshape immune cells and lung tumor immune structures, but did not increase lung cancer growth in a genetically engineered mouse model.

Abstract Introduction Poor CD8 T cell priming is a major barrier to immune control in microsatellite-stable (MSS) colorectal cancer (CRC), resulting in dysfunctional T cells that fail to respond to immune checkpoint blockade (ICB). Nr4a transcription factors are associated with both exhaustion and memory formation–an unresolved paradox with direct relevance to immunotherapy. Methods We used an organoid-derived MSS CRC model with tunable neoantigen expression to compare weak versus strong CD8 T cell priming. Conditional Nr4a1 deletion in CD8 T cells was used to examine early and late responses under both priming conditions, as well as tumor control and survival. Nr4a1 deletion was combined with intradermal (ID) or oral Listeria monocytogenes (Lm-Ova) neoantigen vaccination to evaluate effects on central and tissue-resident memory. Results Weak neoantigen priming rapidly induced CD8 T cell dysfunction accompanied by Nr4a upregulation, whereas strong priming elicited robust effector responses, revealing a priming strength-dependent role of Nr4a. Nr4a1 deletion enhanced early effector responses under both priming conditions but markedly increased tissue-resident memory formation only under weak priming, consistent with a priming context— and T cell stage—specific function of Nr4a1. This contrasts with prior reports that Nr4a1 is important for the development of resident memory T cells. Nr4a1 deletion led to enhanced survival in MSS CRC. Combining Nr4a1 deletion with ID or Lm-Ova vaccination enhanced central and tissue-resident memory, demonstrating that Nr4a1 is dispensable for robust memory formation under certain priming contexts. Conclusion These findings identify a targetable pathway to overcome early CD8 T cell dysfunction induced by suboptimal priming and establish Nr4a1 as a molecular rheostat linking priming strength and T cell fate to immune outcomes. Nr4a1 modulation–particularly in combination with vaccination–offers a promising strategy to enhance antitumor immunity in MSS CRC and other ICB-resistant cancers. Funding Source n/a Topic Categories Vaccines and Immunotherapy (VAC)

Normal pancreas function supports both digestion and the hormonal regulation of whole-body metabolism. We find pancreatic ductal adenocarcinoma (PDAC) disrupts the normal function of the remaining pancreas, leading to altered systemic metabolism and peripheral tissue wasting that begins early in disease progression. Using mouse models of PDAC, we find small pancreas tumors lead to both endocrine and exocrine pancreatic dysfunction that results in systemic nutrient depletion and loss of both muscle and fat tissue. Providing free glucose in the diet that is absorbed despite pancreatic exocrine dysfunction causes hyperglycemia and blunts fat wasting without affecting muscle loss. Muscle mass can be restored by free dietary amino acids or pancreatic enzyme supplementation. Exocrine dysfunction causing reduced dietary protein digestion promotes muscle proteolysis and autophagy. Autophagy is a major driver of muscle wasting in PDAC, as muscle-specific deletion of the core autophagy gene Atg7 also reduces muscle wasting. Disrupting muscle autophagy without restoring systemic nutrition slows tumor growth and improves survival of mice with PDAC. Tracing the fate of amino acids released from muscle of mice with PDAC shows redistribution to both tumor and host tissues. Notably, improving nutrition in mice with disrupted muscle autophagy promotes tumor growth. Together, the data argue that early peripheral tissue wasting associated with early pancreatic cancer is driven by altered normal pancreatic organ function that leads to reduced nutrition and enhanced muscle autophagy, releasing nutrients to support both tumor and host metabolism.

Pancreatic ductal adenocarcinoma (PDAC) frequently recurs and metastasizes despite intensive therapy. The neural-like progenitor (NRP) transcriptional program is enriched in residual disease after neoadjuvant chemotherapy and radiotherapy, but its basis has remained unclear. We hypothesized that NRP represents a regeneration program co-opted by tumors recovering from cytotoxic injury. NRP signatures were strongly enriched in normal pancreatic injury and regeneration, and NRP cancer cells co-expressed transcription factors involved in pancreatic development. Our data support cell-intrinsic contributions and implicate IL-1β-associated inflammatory signaling as a plausible microenvironmental driver of elevated NRP expression. To enable direct phenotypic comparison with other cancer cell states, we established isogenic mouse organoid overexpression models for transcription factors linked to NRP, classical, and basal-like states. Glis3 emerged as a key NRP-associated factor, promoting clonogenicity, tumor growth, and metastasis. These findings identify a clinically relevant developmental regeneration program that emerges in PDAC after treatment.

Tumor progression is driven by dynamic interactions between cancer cells and their surrounding microenvironment. Here we integrate high-resolution spatial transcriptomics and evolving lineage-tracing technologies to elucidate how tumor expansion, plasticity and metastasis co-evolve with microenvironmental remodeling in a Kras;Trp53-driven mouse model of lung adenocarcinoma. We find that subclonal expansion contributes to a hypoxic, immunosuppressive and fibrotic microenvironment that is associated with the emergence of prometastatic cancer cell states. We use tumor phylogeography to delineate intercellular interactions that are rewired in the expanding tumor niche and use co-culture systems to dissect how intercellular interactions and hypoxia influence cancer cell state. Furthermore, we find that metastases arise from spatially confined primary tumor subclones and remodel the distant metastatic niche into a fibrotic, collagen-rich microenvironment. Together, we present a comprehensive dataset integrating spatial assays and lineage tracing to elucidate how sequential changes in cancer cell state and microenvironmental structures cooperate to promote tumor progression.

Regulatory T (Treg) cells suppress immune responses and are considered a major barrier to productive anti-tumor immunity. In this issue of Immunity, Huang et al. report that interleukin (IL)-10-producing Treg cells restrain tumor growth by suppressing IL-17 production in colorectal cancer, highlighting a functional division of labor within the Treg compartment with significance for immunotherapy treatments.

Abstract Introduction Cancer mutations can result in the expression of tumor-specific “neoantigens” that are targeted by T cells. Neoantigen expression drives robust T cell infiltration of tumors across cancer models, yet most of these T cells are not specific for the neoantigens and contain both pathogenic and immunosuppressive subsets. We sought to investigate how neoantigen-specific CD4+ T cells orchestrate nonspecific “Passenger” T cell responses within tumors and determine their impact on tumor progression. Methods Our studies used an autochthonous murine model of lung adenocarcinoma (KrasG12D/+; tp53-/-) in which tumors are synchronously initiated via lentiviral delivery of Cre recombinase with neoantigen, and employed flow cytometry, histological analyses, single-cell transcriptomics, and temporal immune cell perturbations. Results Expression of a single MHC-II-restricted neoantigen (mITGB1) unexpectedly promoted tumor development. mITGB1 expression drove robust intratumoral enrichment of CD4+ and CD8+ Passenger T cells compared to mITGB1-neg controls. A fraction of Passenger T cells in mITGB1+ tumors uniquely displayed hallmarks of antigen experience, clonal expansion, and readily formed aggregates with B cells. Passenger T cells persisted in mITGB1+ tumors for at least three weeks, while lymphocytes in mITGB1-neg tumors rapidly vanished if cells could not traffic from the periphery. Critically, long-term maintenance of Passenger T cells within tumors required continuous expression of the neoantigen. Conclusion Our study reveals an unappreciated role for neoantigen-specific CD4+ T cells in promoting tumorigenesis and actively coordinating non-specific Passenger T cell responses within tumors. Paradoxically, neoantigen expression may furnish the tumor microenvironment with novel T cell specificities and unlock cell states that support tumor growth alongside populations that work to combat progression. Funding Source Jane Coffin Childs Fund, Hope Funds for Cancer Research Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Osteosarcoma (OS) genomes are characterized by complex genomic rearrangements (CGRs) that drive genomic instability and clonal diversification early in tumor evolution. As a result, OS tumors display high inter-patient variability, which has hindered molecular stratification and targeted therapeutic development. To study genomic complexity in OS and credential a genetically engineered mouse model of the disease (Sp7-Cre Trp53fl Rb1fl), we performed high-depth and multi-region whole genome sequencing (WGS) of 35 tumor samples from 24 mice. Similar to human OS, the murine OS tumors (mOS) had a high number of somatic structural variants (158 per tumor) with low tumor mutational burden of single nucleotide variants (0.87 mutations/MB). CGRs were identified in 63% (15/24) of mOS cases, most frequently affecting chromosome 15 (33%, 8/24 mice) and resulting in Myc amplification in 6 mice, ranging from 5 to 104 copies. Myc amplification was verified with DNA FISH, long-read sequencing and gene expression data, which revealed examples of Myc amplification in both extrachromosomal circular DNA (ecDNA) and in derivative chromosomes generated by CGRs. PTEN loss occurred frequently (59% 12/22 mice), and contributed to osteosarcomagenesis, as demonstrated by tumor initiation with in vivo CRISPR/Cas9-mediated deletion experiments (2 mice). Together, these results demonstrate that a preclinical model of osteosarcoma can generate the genomic heterogeneity and complexity of the human disease, thereby facilitating research into mechanisms of tumor initiation and drivers of progression and relapse.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers and shows resistance to any therapeutic strategy used. Here we tested small-molecule inhibitors targeting chromatin regulators as possible therapeutic agents in PDAC. We show that JQ1, an inhibitor of the bromodomain and extraterminal (BET) family of proteins, suppresses PDAC development in mice by inhibiting both MYC activity and inflammatory signals. The histone deacetylase (HDAC) inhibitor SAHA synergizes with JQ1 to augment cell death and more potently suppress advanced PDAC. Finally, using a CRISPR-Cas9-based method for gene editing directly in the mouse adult pancreas, we show that de-repression of p57 (also known as KIP2 or CDKN1C) upon combined BET and HDAC inhibition is required for the induction of combination therapy-induced cell death in PDAC. SAHA is approved for human use, and molecules similar to JQ1 are being tested in clinical trials. Thus, these studies identify a promising epigenetic-based therapeutic strategy that may be rapidly implemented in fatal human tumors.

The pancreas regulates whole-body metabolism through its exocrine and endocrine functions. In pancreatic ductal adenocarcinoma (PDAC), tumor-driven exocrine dysfunction contributes to systemic nutrient depletion, but its role in muscle wasting remains poorly understood. Using genetically engineered mouse models (GEMMs) of PDAC and orthotopic implantation of murine PDAC cells, we observed early disease features including hypoglycemia, elevated circulating branched-chain amino acids, and loss of muscle and fat mass. Skeletal muscle from tumor-bearing mice exhibited suppressed mTORC1 signaling, reduced protein synthesis, activated AMPK signaling, and elevation of Foxo1/Foxo3a-driven lysosome-autophagy pathways, leading to muscle proteolysis. Loss-of pancreatic acinar cells impaired exocrine function, whereas dietary enzyme supplementation rescued protein digestion and restored muscle mass. Muscle-specific deletion of Atg7 reduced muscle wasting, slowed tumor growth, and improved survival. Stable isotope tracing using a 15N-labeled Spirulina diet revealed that amino acids derived from host muscle were utilized by tumors and other tissues. Notably, in mice with impaired muscle autophagy, a high elemental amino acid diet increased mortality, while a low elemental amino acid diet improved survival—suggesting that excess nutrients may fuel tumor growth when muscle catabolism is blocked. Together, these findings indicate that PDAC-induced exocrine insufficiency triggers a starvation-like state that promotes muscle wasting through autophagy, ultimately supporting tumor and host tissue metabolism. Yetiş Gültekin, Sharanya Sivanand, Kian Moritz Eghbalian, Anna Marie Barbeau, Keene Abbott, George Eng, Tori Tavernier, Brian Do, Elif Ozcelik, Sabrina Hu, Tenzin Kunchok, Millenia Waite, Daniel A. Sharygin, Yigit Kaan Kizlier, Will Freed-Pastor, Tyler Jacks Omer Yilmaz, Jonathan Nowak, Brian Wolpin, Matthew G. Vander Heiden. Pancreatic cancer-associated systemic nutrient starvation elevates autophagic proteolysis in the muscle [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A045.

CD4+ T cells can be either protective or pathogenic in cancer [1-5], but how they interact with cancer cells to produce these effects remains poorly understood. Here, we developed a flexible autochthonous platform to introduce CD4+ T cell antigens in combination with CD8+ T cell antigens in a mouse lung cancer model. We found that tumor-specific CD4+ T cells potentiate CD8+ T cell control of tumor progression. Unlike previous studies emphasizing intratumoral interactions among dendritic cells, CD4+ T cells, and CD8+ T cells [4,5], we found that CD4+ T cell function and tumor control depend on direct antigen presentation by cancer cells via major histocompatibility complex class II (MHC-II). This direct interaction between cancer cells and CD4+ T cells led to increased immune infiltration, inflammatory remodeling, and sustained CD8+ T cell effector functions within tumors. Building on this, we demonstrate that tumor vaccination targeting both CD4+ and CD8+ T cell neoantigens significantly reduces tumor burden and requires cancer cell MHC-II presentation. These findings reveal an underappreciated mechanism of CD4+ T cell “help” and suggest a therapeutic approach targeting cancer cell presentation of MHC class II antigens.

Nerves are an integral component of the tumor microenvironment, contributing to cancer progression, metastasis, morbidity, and mortality. In pancreatic ductal adenocarcinoma (PDAC), worse clinical outcomes are associated with perineural invasion (PNI), a process by which cancer cells surround and invade nerves. Here, we employed whole-transcriptome and single-cell spatial transcriptomics to identify candidate tumor-nerve interactions that promote PNI. We discovered that Pdgfd signaling promotes key features of nerve invasion. Mechanistically, Pdgfd stimulated cancer cell invasiveness, neurite outgrowth, and direct physical engagement with glia. Pharmacological blockade of this axis reduced each of these processes in vitro as well as PNI in vivo. Thus, Pdgfd-Pdgfrb signaling mediates PNI by coordinating multifaceted cancer-neuron-glia interactions and represents a promising therapeutic strategy aimed at disrupting harmful cancer-nerve crosstalk.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide, with an increasing incidence and mortality rate among younger patients. Although various risk factors have been identified, they remain insufficient to fully explain the rise of early-onset CRC. While genetic driver mutations are necessary for tumorigenesis, they alone are not sufficient to drive malignant transformation CRC. This is evident as these mutations are often present in histologically normal tissue. A critical unresolved question is: why do some benign polyps progress to malignant CRC while others remain non-invasive? Studying this phenomenon in human patients is challenging, as the earliest stages of benign-to-malignant transition are difficult to capture and cannot be functionally interrogated. To address this, we developed a new genetically engineered mouse model that recapitulates the stepwise-progression of colon cancer by combining in vivo CRISPR-Cas9 editing in the distal colon and an inducible “split-Cre” system. This allows us to decouple tumor initiating events from those that drive malignant progression. Benign adenomas are induced via colonoscopy guided injection of lentivirus expressing single guide RNA against the Apc gene (mutated in ∼80% of human CRC). Recombination of Kras LSL-G12D and Trp53 flox/flox alleles (modeling the most common drivers of advanced CRC in humans) is spatiotemporally controlled via an inducible split Cre-recombinase system, with C-terminal Cre (CreC) delivered in the initiating lentivirus and N-terminal Cre (CreN), fused to a destabilization domain (dd) and Estrogen Receptor T2 (ERT2), expressed from an internal ribosome entry site (IRES) following the stop codon of the endogenous Epcam gene (conferring epithelial specificity). We have validated that this model can capture the full histological spectrum of CRC benign-to-malignant progression: benign adenoma, intramucosal carcinoma, early invasion, and adenocarcinoma-in-adenoma with extensive histological and transcriptional intratumoral heterogeneity. In many of these Apc knockout polyps, we also observe multiple distinct regions with Kras G12D recombination alone and in combination with Trp53 knockout, recapitulating the polyclonal mutational heterogeneity of early CRC in humans. We have performed 10x Visium HD spatial transcriptomics analysis on these samples to elucidate the earliest transcriptional changes associated with stepwise Kras G12D and Trp53 loss-of-function mutations and benign-to-malignant transition. Through comprehensive tumor kinetics and spatial transcriptomic analyses, we seek to uncover deeper insights into the earliest tumor intrinsic and microenvironmental mechanisms underlying CRC benign-to-malignant transition. This approach holds significant potential to inform strategies for CRC early detection and prevention. Yihan Qin, Daniel Zhang, Nikita Persaud, Nischal Bhandari, Zakeria Aminzada, Colin McLaughlin, Song Han, Alex Liu, Rodrigo Romero, Santiago Naranjo, Claire Regan, William Rideout III, Alexander Cicala, Karen Yee, Jonathan Preall, Semir Beyaz, Sepideh Gholami, Zhen Zhao, Tyler Jacks, Peter M. Westcott. Capture the early benign-to-malignant transition of colon cancer in the mouse [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 LB475.

Elevated or ectopic expression of neuronal receptors promotes tumour progression in many cancer types1,2; neuroendocrine (NE) transformation of adenocarcinomas has also been associated with increased aggressiveness3. Whether the defining neuronal feature, namely electrical excitability, exists in cancer cells and impacts cancer progression remains mostly unexplored. Small-cell lung cancer (SCLC) is an archetypal example of a highly aggressive NE cancer and comprises two major distinct subpopulations: NE cells and non-NE cells4,5. Here we show that NE cells, but not non-NE cells, are excitable, and their action potential firing directly promotes SCLC malignancy. However, the resultant high ATP demand leads to an unusual dependency on oxidative phosphorylation in NE cells. This finding contrasts with the properties of most cancer cells reported in the literature, which are non-excitable and rely heavily on aerobic glycolysis. Additionally, we found that non-NE cells metabolically support NE cells, a process akin to the astrocyte-neuron metabolite shuttle6. Finally, we observed drastic changes in the innervation landscape during SCLC progression, which coincided with increased intratumoural heterogeneity and elevated neuronal features in SCLC cells, suggesting an induction of a tumour-autonomous vicious cycle, driven by cancer cell-intrinsic electrical activity, which confers long-term tumorigenic capability and metastatic potential.