Sarver, Aaron L.

Diffuse B-cell lymphomas are the most prevalent canine hematologic malignancies, and their clinical presentation resembles that of human B-cell non-Hodgkin lymphomas (NHLs). However, a lack of a clear subtyping framework perpetuates imprecise treatment approaches that fail to address mechanisms of therapy resistance. Here, we employed deep phenotyping via serial sampling and longitudinal transcriptomics to evaluate intratumoral composition and therapy response in a 6-year-old neutered male goldendoodle with stage 5A marginal zone lymphoma (MZL). Following sequential treatment consisting of a single IV dose of oncolytic vesicular stomatitis virotherapy (VSV) and standard CHOP chemotherapy 30 days later, we performed scRNAseq on seven serial lymph node biopsies and spatial sequencing on a resection taken 10 days post-VSV treatment. B cells (>90%) comprised three transcriptionally distinct and temporally stable subpopulations, with specific copy number profiles and robust spatial organization despite disrupted lymph node architecture. Analysis across subpopulations revealed downregulation of the quiescence program including KLF2, suggesting similarity to KLF2-deficient human MZL. While VSV successfully reached target B cells, downstream transcriptional responses were predominantly localized to the T-cell compartment. T cells (≤6%) showed increased proliferation and upregulation of cytotoxicity markers post-VSV administration, enriching for leukocyte-mediated cytotoxicity pathways consistent with an anti-viral immune response. Ultimately, these results provide an in vivo proof-of-concept for the treatment paradigm in canine lymphoma, while emphasizing the need for improved subtyping frameworks and multidimensional treatment strategies targeting intratumoral heterogeneity.

Abstract Glioblastoma (GBM) remains a deadly cancer driven in part by invasion of tumor cells into the brain. Transcriptomic analyses have identified distinct molecular subtypes, but mechanistic differences that account for clinical differences are not clear. In this study, we show that, as predicted by the motor-clutch model of cell migration, mesenchymal glioma cells are more spread, generate larger traction forces, and migrate faster in brain tissue compared with proneural cells. Despite their rapid migration and comparable proliferation rates in vitro, mice with mesenchymal tumors survive longer than those with proneural tumors. This improved survival correlated with an immune response in mesenchymal tumors, including T cell–mediated. Consistently, inducing mesenchymal tumors in immunodeficient mice resulted in shorter survival, supporting a protective immune role in mesenchymal tumors. Thus, mesenchymal tumors have aggressive migration but are immunologically “hot,” which suppresses net proliferation. These two features counteract each other and may explain the lack of a strong survival difference between subtypes clinically, while also opening up new opportunities for subtype-specific therapies. Significance: This study highlights new mechanical and immunologic insights into GBM molecular subtypes using an integrated modeling–genome engineering strategy, which can potentially facilitate GBM subtype-specific therapeutic strategies.

Abstract Background: Canine nodal marginal zone lymphoma (MZL) is the second most common histologic diagnosis of diffuse B-cell lymphomas in dogs, but a molecular profile that can distinguish this condition from the more common canine diffuse large B-cell lymphoma (DLBCL) has been elusive. Here, we use single-cell sequencing to define the transcriptomic signatures of canine MZL before and after oncolytic virotherapy followed by traditional chemotherapy. Objective: The aims of this study were to define the canine MZL transcriptome at single cell resolution and to evaluate its changes in the face of selective pressures introduced by therapy. Methods: Longitudinal biopsies and fine needle aspirates were collected from a dog with MZL enrolled in an immunotherapy trial. Peripheral blood B-cells from healthy dogs served as reference controls. Single-cell sequencing was done using the 10x Genomics Chromium GEM X platform (v3.1), and data was aligned to the canFam4_Y genome. Quality control, clustering, and cell annotation were performed using Seurat in R (v4.4.0). Differential gene expression analysis (DGEA) was performed on 500 randomized MZL B-cells and 500 healthy canine B-cells. InferCNV was deployed on the longitudinal MZL samples for prediction of tumor B-cell CNV profiles. Results: When compared to normal peripheral blood B-cells, the tumor cells were characterized by downregulation of quiescence-associated genes, including KLF2. Copy number analysis uncovered loss on canine chromosome 20 (CFA20) encoding KLF2, gain on CFA13, and gains on CFA32 and CFA36. While an anti-viral T-cell response was identified after VSV treatment, the B-cell subpopulations retained stable transcriptomes, with a disappearance of proliferating B-cells after chemotherapy. Conclusion: This is the first report describing the molecular properties of canine MZL at single cell resolution. Clonal diversity was apparent in the tumor, with copy number loss on CFA20, presumably leading to loss of KLF2. The transcriptional features of the tumor cells remained stable even in the face of a T-cell response to an oncolytic virus. Taken together, the results document silencing of KLF2 as a key phenotypic homology between canine and human MZL, supporting the potential for comparative approaches to enhance our understanding of the significance of KLF2 silencing in this disease. Citation Format: Elise Weir, Grace Walker, Jaime F. Modiano, Aaron L. Sarver, Shruthi Naik, Amy Treeful. Reduced KLF2 expression in canine marginal zone lymphoma mirrors human disease [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A043.

Abstract Introduction: Lymphomas are among the most common malignancies in dogs, and their morphologic and biological similarities to human counterparts have established them as widely accepted models of human disease. However, emerging molecular data suggest that conservation varies by subtype, and that many common lymphoma subtypes are principally convergent rather than homologous conditions, requiring a critical reassessment of when and how canine lymphomas provide relevant models of human disease. Methods: We employed conventional pathology, immunohistochemistry, and next generation sequencing platforms, including single-cell sequencing and spatial genomics, to characterize the transcriptional landscape and cellular organization of canine lymphomas, complemented by population metadata. Results: Our analysis revealed that canine DLBCL lacks the traditional GCB/ABC bifurcation seen in humans. While activation of NFκB pathways is seen commonly in these tumors, a germinal center-like DLBCL subtype is not apparent in dogs, and the transcriptional signatures are distinct from those of activated B-cell like DLBCL. Instead, we identified a novel canine-specific DLBCL subset driven by an AP-1-associated transcriptional signature, mirroring advanced human follicular lymphoma rather than human DLBCL. In contrast, emerging data suggest that downregulation of quiescence programs might be a shared feature of both human and canine MZLs, making this subtype a candidate for conserved disease mechanisms. While canine T-cell lymphomas exhibited extensive transcriptional heterogeneity, the samples in our dataset did not align with established human T-cell subgroups, suggesting that high incidence in dogs might not equate to molecular similarity. Conclusions: Our results suggest canine and human lymphomas are principally convergent rather than homologous diseases whose evolution and topological organization are constrained by developmental programs. The distinct transcriptional programs of canine and human DLBCLs reveal informative biological divergence, while potential shared features of MZL suggest contexts where comparative oncology may illuminate conserved disease mechanisms. Canine T-cell lymphomas exhibit extensive transcriptional heterogeneity that, at present, defies simple classification, mirroring challenges in their human counterparts and underscoring the need for larger studies to establish molecular patterns that inform causation, prognosis, and treatment. These findings highlight contexts where distinct and conserved molecular features between canine and human lymphoma can reveal shared and divergent disease mechanisms, ultimately underscoring the need for subtype-specific validation as canine lymphomas are adopted as models of human disease. Use of AI: Generative AI was used to improve clarity and readability of the abstract. AI was not used to conduct the study or to produce new text or material. Citation Format: Jaime F. Modiano, Amy E . Treeful, Grace E. Walker, Davis M. Seelig, Caitlyn M. Callaghan, Timothy D. O'Brien, Shruthi Naik, Aaron L. Sarver. Different paths to the same destination: Molecular divergence and functional convergence in human and canine lymphomas [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A023.

Abstract Glioblastoma remains a deadly cancer driven in part by invasion of tumor cells into the brain. Transcriptomic analyses have identified distinct molecular subtypes, but mechanistic differences that account for clinical differences are not clear. Here, we show that, as predicted by the motor-clutch model of cell migration, mesenchymal glioma cells are more spread, generate larger traction forces, and migrate faster in brain tissue compared to proneural cells. Despite their rapid migration and comparable proliferation rates in vitro, mice with mesenchymal tumors survive longer than those with proneural tumors. This improved survival correlated with an immune response in the mesenchymal tumors, including T cell-mediated. Consistently, inducing mesenchymal tumors in immunodeficient mice resulted in shorter survival supporting a protective immune role in mesenchymal tumors. Thus, mesenchymal tumors have aggressive migration, but are immunologically ‘hot’ which suppresses net proliferation. These two features counteract each other and may explain the lack of a strong survival difference between subtypes clinically, while also opening up new opportunities for subtype-specific therapies. Significant Statement This study highlights new mechanical and immunological insights into glioblastoma molecular subtypes using an integrated modeling-genome engineering strategy, which can potentially facilitate glioblastoma subtype-specific therapeutic strategies.

Abstract The goal of this study was to evaluate safety, efficacy, and immunological activity of Vesicular Stomatitis Virus (VSV) expressing a species relevant (canine) interferon-beta (IFN-β) and the sodium iodide symporter (NIS), henceforth called VSV, in companion dogs with naturally occurring appendicular osteosarcoma. The hypothesis was that VSV would track to the tumor, induce oncolysis, and lead to enhanced infiltration of immune cells, expansion of virus- and tumor specific T-cell clones, and improved survival outcomes.The first 15 dogs enrolled in the VSV Immunotherapy and Genomics of Osteosarcoma Research (VIGOR) study received a single iv dose of VSV (1x109 TCID50/kg) 10 days before amputation and adjuvant carboplatin chemotherapy. The final 13 dogs were randomized in a double-blinded fashion to receive either a single dose of VSV or placebo. Outcome data were compared to those from an historical control group of 57 dogs with appendicular osteosarcoma treated between 2006 and 2018 using the standard of care of alone. VSV treatment was associated with mild systemic effects (fever and inflammation), which were self-limiting and caused no symptomatology. Alterations in the tumor microenvironment (micronecrosis, increased fibrosis, and increased inflammation) were more common and more pronounced in VSV treated dogs than in placebo-treated dogs.Reduction in the expression of co-regulated cell cycle gene clusters in tumors after VSV-treatment was observed in dogs with longer survival. Conversely, increased expression of co-regulated immune/inflammatory gene clusters was observed in both VSV-treated dogs and in placebo-treated dogs, suggesting this might be, at least partly, due to the biopsy procedures. Assessment of changes in the clonal composition of T cells within the tumors, draining lymph nodes, and systemic circulation is in progress. No differences were evident in median event-free or overall survival times among the different groups, although aspects of the study design and sample size could obscure differences. Intriguingly, a “tail” of nine dogs (41%) that were long-term survivors (>450 days) was apparent in the VSV-treated group. All but one of these long-term survivors had evidence of inflammation in the pre-treatment tumor samples or showed increased inflammation in the post-treatment samples. We conclude that neoadjuvant VSV has an excellent safety profile and provides a survival benefit for a subset of dogs with spontaneous bone cancer. Ongoing work seeks to establish distinguishing characteristics that identify those individuals most likely to benefit from this therapy, as well as to evaluate the potential to enhance this benefit through the addition of novel immunomodulatory agents as part of our efforts to translate this approach to human cancer patients. Citation Format: Kelly M. Makielski, Aaron L. Sarver, Aishwarya Sathyanarayan, Michael S. Henson, Kathleen M. Stuebner, Alexandru-Flaviu Tabaran, Ingrid Cornax, Gerard O'Sullivan, Andrea Chehadeh, Donna Groschen, Kelly Bergsrud, Lauren J. Mills, Amber L. Winter, Sara Pracht, Milcah C. Scott, Michael A. Farrar, Gary R. Cutter, Joseph S. Koopmeiners, Stephen J. Russell, Shruthi Naik, Jaime F. Modiano. Oncolytic Vesicular Stomatitis Virus expressing a species relevant interferon-beta is safe and provides a survival benefit when added as neoadjuvant to the standard of care for dogs with naturally occurring bone cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1747.

Abstract In Glioblastoma (GBM), tumor spreading is driven by tumor cells’ ability to infiltrate healthy brain parenchyma, which prevents complete surgical resection and contributes to tumor recurrence. GBM molecular subtypes, classical, proneural and mesenchymal, were shown to strongly correlate with specific genetic alterations (Mesenchymal: NF1; Classical: EGFRVIII; Proneural: PDGFRA). Here we tested the hypothesis that a key mechanistic difference between GBM molecular subtypes is that proneural cells are slow migrating and mesenchymal cells are fast migrating. Using Sleeping Beauty transposon system, immune-competent murine brain tumors were induced by SV40-LgT antigen in combination with either NRASG12V (NRAS) or PDGFB (PDGF) overexpression. Cross-species transcriptomic analysis revealed NRAS and PDGF-driven tumors correlate with human mesenchymal and proneural GBM, respectively. Similar to human GBM, CD44 expression was higher in NRAS tumors and, consistent with migration simulations of varying CD44 levels, ex vivo brain slice live imaging showed NRAS tumors cells migrate faster than PDGF tumors cells (random motility coefficient = 30µm2/hr vs. 2.5µm2/hr, p < 0.001). Consistent with CD44 function as an adhesion molecule, migration phenotype was independent of the tumor microenvironment. NRAS and human PDX/MES tumor cells were found to migrate faster and have larger cell spread area than PDGF and human PDX/PN tumors cells, respectively, in healthy mouse brain slices. Furthermore, traction force microscopy revealed NRAS tumor cells generate larger traction forces than PDGF tumors cells which further supports our theoretical mechanism driving glioma migration. Despite increased migration, NRAS cohort had better survival than PDGF which was attributed to enhanced antitumoral immune response in NRAS tumors, consistent with increased immune cell infiltration found in human mesenchymal GBM. Overall our work identified a potentially actionable difference in migration mechanics between GBM subtypes and establishes an integrated biophysical modeling and experimental approach to mechanically parameterize and simulate distinct molecular subtypes in preclinical models of cancer.

Osteosarcoma is the most common primary tumor of bone. Osteosarcomas are rare in humans, but occur more commonly in dogs. A comparative approach to studying osteosarcoma has highlighted many clinical and biologic aspects of the disease that are similar between dogs and humans; however, important species-specific differences are becoming increasingly recognized. In this review, we describe risk factors for the development of osteosarcoma in dogs and humans, including height and body size, genetics, and conditions that increase turnover of bone-forming cells, underscoring the concept that stochastic mutational events associated with cellular replication are likely to be the major molecular drivers of this disease. We also discuss adaptive, cancer-protective traits that have evolved in large, long-lived mammals, and how increasing size and longevity in the absence of natural selection can account for the elevated bone cancer risk in modern domestic dogs.

Abstract Medulloblastoma and central nervous system primitive neuroectodermal tumors (CNS-PNET) are aggressive, poorly differentiated brain tumors with limited effective therapies. Using Sleeping Beauty (SB) transposon mutagenesis, we identified novel genetic drivers of medulloblastoma and CNS-PNET. Cross-species gene expression analyses classified SB-driven tumors into distinct medulloblastoma and CNS-PNET subgroups, indicating they resemble human Sonic hedgehog and group 3 and 4 medulloblastoma and CNS neuroblastoma with FOXR2 activation. This represents the first genetically induced mouse model of CNS-PNET and a rare model of group 3 and 4 medulloblastoma. We identified several putative proto-oncogenes including Arhgap36, Megf10, and Foxr2. Genetic manipulation of these genes demonstrated a robust impact on tumorigenesis in vitro and in vivo. We also determined that FOXR2 interacts with N-MYC, increases C-MYC protein stability, and activates FAK/SRC signaling. Altogether, our study identified several promising therapeutic targets in medulloblastoma and CNS-PNET. Significance: A transposon-induced mouse model identifies several novel genetic drivers and potential therapeutic targets in medulloblastoma and CNS-PNET.