Wang, Panwen

Abstract Introduction The underlying immunopathogenesis of inflammatory arthritis (IA) immune-related adverse event (irAE) remains obscure. Unlike rheumatoid arthritis (RA), where autoantibodies and B cell dysfunction are central features, the contribution of humoral immunity to IA-irAE is unclear. Methods Here, we performed immunophenotyping of peripheral blood from IA-irAE patients, and compared them with seronegative RA patients, ICI-treated patients without irAE, and healthy controls. Results IA-irAE was marked with increased cytotoxic gene expression and metabolic activation in T cells, and reduced CXCR3 and CCR6 expression in CD4+ T cells. Contrary to seronegative RA, IA-irAE patients displayed no significant elevation in autoantibody levels or atypical CD11c+CD21⁻ B cells. IA-irAE was further characterized by elevated levels of IL-6, IL-12, and type I IFN, which correlated with the T cell activation phenotypes. Conclusion Altogether, our findings define IA-irAE as a disease with certain immunological features distinctive from RA, representing a potentially T cell driven, autoantibody-independent autoimmunity. These results offer insights into immune tolerance breakdown and therapeutic targeting in irAEs. Funding Source National Institute of Arthritis and Musculoskeletal and Skin Diseases Mark E. and Mary A. Davis Initiative in Rheumatoid Arthritis Research Mayo Foundation for Medical Education and Research Topic Categories Basic Autoimmunity (BA)

ABSTRACT Germline variation shapes cancer risk, yet its influence on the evolutionary dynamics of established tumors remains poorly understood. In multiple myeloma, subclonal diversification drives disease progression and treatment failure, but the heritable factors that modulate this process are unknown. Here, we show that germline variation is associated with tumor evolutionary features, implicating inherited regulation in subclonal expansion. Integrating germline variation with tumor evolutionary parameters identifies variants associated with evolutionary features, with signals enriched in regulatory regions, consistent with a transcriptional basis. We further identify TBKBP1 as a key locus linking germline variation to tumor evolution and clinical outcome. Germline variation at this locus is associated with TBKBP1 expression and subclonal expansion, and TBKBP1 expression correlates with adverse prognosis, consistent across independent cohorts. Functional analyses demonstrate that TBKBP1 promotes proliferation and activates MYC, mTORC1 and non-canonical NF-κB signaling pathway. Together, these findings establish germline regulatory variation as a determinant of tumor evolutionary dynamics and identify TBKBP1 as a mediator linking inherited variation to subclonal expansion and disease progression in multiple myeloma.

Abstract Expression quantitative trait locus (eQTL) mapping is used to identify the functional link between a genomic variant and a gene’s expression. A significant eQTL association does not mean a causal relationship or mechanism, and further investigation is needed to understand how a single-nucleotide polymorphism (SNP) impacts gene expression. One of the most plausible explanations for eQTL is that a genomic variant affects transcription factor (TF) binding and thus impacts its regulation on target genes (TGs). However, the current eQTL model does not provide information on the TF and how its regulation is mediated by the SNP’s genotypes. Here, we propose a new method called differential co-expression QTL (ceQTL) among different alleles using Chow statistics to specifically detect eQTLs that are bound by a particular TF. We start with building a trio of TF, its TG, and related SNP, and then test the significant coefficient difference among different genotypes of the SNP. We applied this ceQTL model to simulated data and the lung tissue datasets from the genotype-tissue expression project. The simulated data results showed that the model was robust to detect true ceQTLs at variable sample sizes and different minor allele frequencies as measured by area under the curve. Our tool also performed a TF binding affinity analysis to add another layer of evidence for functional interpretation. In summary, ceQTL analysis provides a more interpretable and biological insight into the mechanism of eQTL and transcriptomic regulation, which would help us better understand how genomic variants affect phenotypes and diseases.

Abstract Background: Hepatocellular carcinoma (HCC) is largely refractory to immune checkpoint blockade due to immune exclusion, stromal barriers, and aberrant vasculature. We evaluated whether multimodal therapy - external beam radiotherapy (EBRT) for antigen release, intratumoral dendritic cell (DC) vaccination for antigen presentation, PD-L1 blockade for immune activation, and VEGF inhibition for vascular normalization - could convert “cold” HCC tumors into sustained immune-inflamed states. Methods: Serial biopsies (baseline, post-EBRT, end-of-treatment [EOT], and progression [PD] when available) from three patients enrolled in an ongoing phase II trial (NCT03942328) underwent multiplex immunofluorescence, spatial neighborhood analysis, and transcriptomic profiling. Longitudinal changes in cellular composition, immune-stromal architecture, spatial contact networks, and pathway activity were compared. Results: Baseline tumor microenvironments (TMEs) were strongly immune-excluded, dominated by tumor (PanCK+60-70%) and stroma (SMA+/Vimentin+25-30%) with sparse immune infiltration (<10-15%). Post-therapy, immune complexity and spatial connectivity increased 2-3-fold in all patients. Patient 1 showed robust cold-to-hot conversion with CD8+(3x), CD4+(2.2x), and DC (2.3x) expansion, increased CD8-DC interactions (2.5x), and reduced tumor fraction (-40%). Transcriptomics showed marked upregulation of IFN-γ (↑3.4x), HLA-DRA (↑2.7x), and reductions in VEGFR2 (↓50%) and HIF1A (↓40%), consistent with vascular normalization. Patient 2 exhibited stromal/vascular remodeling and improved T-cell infiltration but restricted immune diversity, with CD4+/CD8+ dyads (↑2.2x) and B/NK cells(↓ 60%). Neighborhood diversity rose (3→7 communities) and immune-tumor proximity improved (distance ↓ 35%). Patient 3 showed transient inflaming with CD8+(↑3x), DC(↑2x), and new CD4-CD20/CD4-CD56 interactions, followed by relapse marked by stromal/myeloid re-expansion (SMA+↑1.8x, CD14+↑2.5x), angiogenic resurgence (VEGFR2/CD34+↑2.2x), contraction of immune networks, and genomic shift from activating CTNNB1 mutation loss to ATM gain. DCs accumulated but localized mainly to DC-DC or DC-myeloid niches, indicating non-productive antigen presentation. Conclusions: Multimodal EBRT+DC+PD-L1/VEGF blockade induces four-sided synergistic TME reprogramming - antigen release, immune priming, checkpoint release, and vascular normalization - promoting cold-to-hot conversion in HCC. Durable inflaming correlates with sustained T-cell/DC expansion and vascular remodeling, whereas relapse reflects stromal/myeloid reinforcement and genomic adaptation. These findings define mechanistic trajectories of TME remodeling and highlight actionable barriers to sustained immunotherapy response in HCC. Citation Format: Lionel Aurelien Kankeu Fonkoua, Christopher L. Hallemeier, Basim Salem, Arina Tkachuk, Kaitlynn McCay, Panwen Wang, Ying Li, Thomas D. Atwell, Krishan R. Jethwa, Caitlin Conboy, Nguyen H. Tran, Leslie A. Washburn, Melody Wu, Chen Wu, Andre De Menezes Silva Corraes, Rondell P. Graham, Jose Caetano Villasboas, Kevin Regan, Zuoyi Shao, Rayaan Kamal, Nathan R. Foster, Haidong Dong, Sean Park, Yi Lin, Lewis R. Roberts. Multimodal immunotherapy remodels the tumor microenvironment in hepatocellular carcinoma: Integrative spatial and transcriptomic profiling from a Phase II trial [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 7935.

Abstract Introduction: Liver cancer remains a leading cause of cancer mortality globally with limited treatment options for patients (pts) with unresectable hepatocellular carcinoma [HCC] and intrahepatic cholangiocarcinoma [iCCA][1,2,3,4]. We hypothesize that combining the intratumoral dendritic cell (DC) vaccination after external beam radiotherapy (EBRT) without (phase I) and with (phase II) systemic atezolizumab and bevacizumab (atezo/bev for HCC) would enhance tumor-specific immunity and improve clinical outcomes, and present preliminary results from our phase I/II trial (NCT03942328). Methods: Peripheral blood mononuclear cells (PBMCs) were collected at baseline (PRE), after radiation (EBRT), and after completion of DC without or with atezo/bev (POST). Single-cell RNA sequencing with TCR sequencing were analyzed using CellRanger v7.0.1, Immunopipe, Seurat v5.3.0, & Gene-set enrichment analysis (GSEA). Long EFS (EFS-L) is defined as >=12 months (mo) for phase I and >=18mo for phase II, and short EFS (EFS-S) below that. Results: Seventeen patients have been analyzed to date (Phase I: n=8 [4 HCC, 4 iCCA]; Phase II: n=9 HCC). 674,106 cells were sequenced (146,843 PRE; 168,838 EBRT; 114,437 POST). GSEA for EFS-L vs EFS-S were comparable at PRE, EBRT and POST between phase I and phase II. EFS-S was associated with enrichment of interferon alpha (IFNα; B naïve, monocytes, pDC, cDC2, NK, Treg, CD8 Tcm and Tem at PRE and POST; B intermediate, CD4 Tem and NK at EBRT) and reactive oxygen species (ROS; PRE: pDC, CD8 Tcm, Tem. EBRT: CD4 Tem. POST: CD8 T naïve Tem, CD4 Tcm & Tem, cDC2) pathways. In contrast, EFS-L was associated with enrichment of WNT-β-catenin (WNT; CD14 mono at EBRT & POST, B intermediate at EBRT), TGFb (PRE: NK; EBRT: B intermed, CD4 Tem; POST: monocytes, CD4 naïve & Tem, pDC, cDC2), and angiogenesis (EBRT: NK; POST: monocytes, cDC2) pathways. Interestingly, enrichment of protein secretion pathway in NK cells was associated with EFS-L, but among other cells were associated with EFS-S (PRE: CD16 mono, Treg; POST: CD16 mono, CD4 & D8 Tem). Compared to phase I, addition of atezo/bev in phase II pts was associated with a significant increase in Gini coefficient (p<0.0001), suggesting expansion of certain TCR clones, along with a trend for increased Gini coefficient from Pre to Post in both phase I and II. Conclusions: Preliminary analysis of this study combining EBRT with intratumoral DC vaccination +/- PD-L1/VEGF blockade identified distinct treatment-related changes in systemic cellular immune profiles. Enhanced TCR clonal diversity in phase II suggests added immunologic benefit from PD-L1/VEGF blockade. As accrual continues, integrated analyses will identify systemic immune correlates of clinical benefit and resistance to this multimodal immunotherapy approach. Citation Format: Melody Wu, Panwen Wang, Chen Wu, Ying Li, Christopher L. Hallemeier, Thomas D. Atwell, Nguyen H. Tran, Andre de Menezes Silva Corraes, Kevin Regan, Zuoyi Shao, Rayaan Kamal, Haidong Dong, Lewis R. Roberts, Sean Park, Yi Lin, Lionel Aurelien Kankeu Fonkoua. Radiation-dendritic cell combination therapy drives systemic cellular immune shifts in liver 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 3741.

Abstract Chimeric antigen receptor (CAR) T-cell therapy achieves high response rates in relapsed/refractory aggressive B-cell non-Hodgkin lymphoma, yet only 40% of patients achieve durable remission. Identifying biological programs linked to long-term response remains critical. As metabolic fitness underlies T-cell persistence and effector function, we profiled metabolic pathways associated with clinical outcomes following commercial CD19 CAR T-cell therapy. Single-cell RNA sequencing of peripheral blood mononuclear cells collected at baseline (BL), peak CAR-T expansion (PK), and one-month post-infusion (M1) was analyzed using CellRanger v7.0.1, immunopipe. Seurat v4.3.0 was applied for unsupervised clustering to delineate cell subsets based on top differentially expressed genes. Patients were categorized as durable complete remission (CR ≥6 months; n = 16), primary refractory (PD1; n = 4), or relapse after initial response (PD2; n = 12). Gene-set enrichment analysis defined metabolic pathway activity across T-cell, monocyte, dendritic cell (DC), and natural killer (NK) subsets. Oxidative phosphorylation (OXPHOS) emerged as the dominant metabolic program distinguishing clinical outcomes. At BL and PK, OXPHOS was consistently enriched in PD1 relative to CR across T-cell, monocyte, and DC subsets, suggesting early oxidative activation in non-responders. By M1, this pattern inverted, with higher OXPHOS activity in CR. PD2 largely paralleled PD1, but several subsets including CD8 T central memory, classical monocytes TGFβ, intermediate monocytes CD38, monocytic myeloid-derived suppressor cell (mMDSC) HIF1A, and mMDSC SIRPA, showed OXPHOS enrichment in CR at BL and/or PK that persisted through M1. In contrast, DC and NK subsets exhibited the opposite pattern: OXPHOS was enriched in CR at PK (conventional DC 2 (cDC2), plasmacytoid DC (pDC), NK, proliferating NK), but shifted toward enrichment in PD2 at M1 (cDC2, NK, NK CD56bright). Glycolysis (GLY) followed a similar trajectory in monocytes and NK cells, with enrichment in PD1/PD2 at BL and PK, followed by enrichment in CR at M1. In T-cells, PD1 maintained GLY enrichment from BL through PK, with no significant differences observed at M1. The inositol-phosphate metabolism pathway showed a more static pattern and was consistently enriched in multiple PD2 effector and memory T-cell subsets without reversal at M1. Taken together, these data reveal distinct, lineage-specific metabolic signatures that differentiate durable remission from early and late progression. Dynamic OXPHOS and GLY programming, characterized by lower activity early and enhanced activity at M1 in CR, may reflect adaptive metabolic programming that supports sustained antitumor immunity. Immune-metabolic profiling may therefore serve as a biomarker of response and highlight actionable metabolic pathways to enhance CAR T-cell durability. Citation Format: Melinda S.Y. Tan, Panwen Wang, Patrizia Mondello, Jacqueline Turner, Andre de Menezes Silva Corraes, Chen Wu, Zuoyi Shao, Kevin Regan, Ma Audrey, Arushi Khurana, Nora N. Benanni, Yucai Wang, Paul Hampel, Jonas Paludo, Saad J. Kenderian, Urshila Durani, Patrick B. Johnston, Jose Caetano Villasboas, Stephen M. Ansell, Ying Li, Haidong Dong, Hu Zeng, Yi Lin. Metabolic pathway signatures defining response to CD19 CAR T-Cell therapy in aggressive B-cell non-hodgkin lymphoma [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 3278.

Abstract Introduction: Therapeutic outcomes in B-cell non-Hodgkin’s lymphoma (B-NHL) have substantially improved with the introduction of chimeric antigen receptor T-cell (CAR-T) therapy. Yet, persistent immune dysfunction and underlying metabolic dysregulation remain major obstacles to achieving durable clinical responses in B-NHL. We hypothesize that mitochondrial pathways play a critical role in mediating anti-tumor immunity during CAR-T therapy. Methods: Peripheral blood mononuclear cells were collected from healthy controls (CNTRL, n=5) and patients with advanced stage lymphoma (LYM) who received FDA approved CAR-T (n=32). Samples were collected before lymphodepletion (BL), at peak CAR-T expansion (PK) and one-month post-infusion (M1). Single-cell RNA sequencing was used to interrogate differential mitochondrial gene expression. Samples were obtained from LYM patients with complete remission for >6 months (CR), primary refractory (PD1), or relapsed (PD2) disease. Results: Compared to CNTRLs, LYM patients exhibited reduced expression of cytochrome c oxidase. At BL, CR patients demonstrated broader mitochondrial differential gene expression across all T-cell subsets relative to PD1 and PD2 and showed higher expression of MT-ATP-6, MT-ATP-8, and MT-CO3. Following CAR-T infusion, global mitochondrial gene expression increased across patients corresponding with expansion of CD8 effector T cells (Tem tumor circulating) and activated peripheral memory T cells (Tpm). Certain mitochondrial genes, such as MT-CO1, were durably expressed across LYM patients at BL, PK, and M1 in peripheral and central memory T cells (Tpm, Tcm). At PK, cytochrome c oxidase and ATP synthase genes were upregulated in CR Tpm and Tcm. At M1, MT-ATP-6, MT-ATP-8, MT-CO3 were significantly upregulated in CD8 memory precursor effector T-cells (MPECs) of CR patients compared to both PD1 and PD2. While CR and PD2 patients appeared clinically responsive at M1, CR samples maintained significantly higher MT-ATP-8 expression across T cell subsets (CD8 MPECS, Tpm, Tcm) compared to PD2 (p values 0.008; <0.0001; <0.0001). Conclusions: We report a temporal shift in immunometabolism with CAR-T therapy in LYM. Clinical responders exhibited sustained mitochondrial activity characterized by persistent expression of ATP synthase in CD8 MPECs and cytochrome c oxidase in Tpm and Tcm. In sum, mitochondrial programming may distinguish effective versus dysfunctional T cells and identify targets for therapeutic development. Citation Format: Jacqueline Turner, Panwen Wang, Patrizia Mondello, Melinda Tan, Christoph Schaefers, Chen Wu, Andre de Menezes Silva Corraes, Kevin Regan, Zuoyi Shao, Ma Audrey, Arushi Khurana, Nora N. Benanni, Yucai Wang, Paul Hampel, Saad J. Kenderian, Jonas Paludo, Urshila Durani, Patrick B. Johnston, Jose Caetano Villasboas, Stephen M. Ansell, Haidong Dong, Ying Li, Zeng Hu, Yi Lin. Mitochondrial transcriptome profiles associated with clinical responses to CAR-T therapy in aggressive lymphoma [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 1213.

The underlying immunopathogenesis of inflammatory arthritis (IA) immune-related adverse event (irAE) remains obscure. Unlike rheumatoid arthritis (RA), where autoantibodies and B cell dysfunction are central features, the contribution of humoral immunity to IA-irAE is unclear. Here, we performed immunophenotyping of peripheral blood from patients with IA-irAE and compared them with patients with seronegative RA, immune checkpoint inhibition–treated patients without irAE, and healthy controls. IA-irAE was marked with increased cytotoxic gene expression and metabolic activation in T cells and reduced CXCR3 and CCR6 expression in CD4 + T cells. Contrary to seronegative RA, patients with IA-irAE displayed no substantial elevation in autoantibody levels or atypical CD11c + CD21 − B cells. IA-irAE was further characterized by elevated levels of interleukin-6 (IL-6), IL-12, and type I interferon, which correlated with the T cell activation phenotypes. Together, our findings define IA-irAE as a disease with certain immunological features distinctive from RA, representing a potentially T cell–driven, autoantibody-independent autoimmunity. These results offer insights into immune tolerance breakdown and therapeutic targeting in irAEs.

Obesity is a persistent public health issue, often resulting in metabolic complications such as insulin resistance (IR). The secretion of pro-inflammatory cytokines from adipose tissue (AT) is increased during obesity, contributing to the impairment of systemic insulin sensitivity. While interventions in animal models have shown that reducing inflammation restores insulin sensitivity, human studies reducing systemic inflammation have produced inconsistent results. We recently demonstrated that three months of high-dose (4 g/daily) ω-3PUFA (fish oil, FO) supplementation improved insulin sensitivity, and decreased systemic and AT inflammation in individuals with obesity (BMI ≥ 30 kg/m2). Given recent studies highlighting the involvement of microRNA (miRNA) in inflammatory cytokine production, we investigated the effect of ω-3PUFA supplementation on AT miRNA expression in this cohort. AT biopsies were collected before and after ω-3PUFA supplementation. miRNA was processed on the Affymetrix miRNA 4.0 GeneChip and analyzed using existing inflammatory gene sets sourced from MSigDB. Unbiased, differentially expressed miRNA analysis identified miR-4498 and miR-5689 as significantly increased after three months of ω-3PUFA supplementation. Real-time PCR confirmed bioinformatic analysis findings. Our study reports the modulation of miRNA in AT, reductions in systemic and AT markers of inflammation, and the improvement of IR post ω-3PUFA supplementation. Further research is needed to elucidate the link between miR-4498, miR-5689, and whole-body insulin sensitivity.

Abstract Motivation Intratumor heterogeneity arises from ongoing somatic evolution and complicates cancer diagnosis, prognosis, and treatment. Reconstructing evolutionary dynamics typically requires spatiotemporal samples, which are often unavailable in clinical settings. Computational approaches that can infer tumor evolutionary history from single-timepoint bulk sequencing data remain limited. Results We present estimating evolutionary events through single-timepoint sequencing (TEATIME), a novel computational framework that models tumors as mixtures of two competing cell populations: an ancestral clone with baseline fitness and a derived subclone with elevated fitness. Using cross-sectional bulk sequencing data, TEATIME estimates mutation rates, timing of subclone emergence, relative fitness, and number of generations of growth. To quantify intratumor fitness asymmetries, we introduce a novel metric—fitness diversity—which captures the imbalance between competing cell populations and serves as a measure of functional intratumor heterogeneity. Applying TEATIME to 33 tumor types from The Cancer Genome Atlas, we revealed divergent as well as convergent evolutionary patterns. Notably, we found that immune-hot microenvironments constraint subclonal expansion and limit fitness diversity. Moreover, we detected temporal dependencies in mutation acquisition, where early driver mutations in ancestral clones epistatically shape the fitness landscape, predisposing specific subclones to selective advantages. These findings underscore the importance of intratumor competition and tumor-microenvironment interactions in shaping evolutionary trajectories, driving intratumor heterogeneity. Lastly, we demonstrate that TEATIME-derived evolutionary parameters and fitness diversity offer novel prognostic insights across multiple cancer types. Availability and implementation R implementation of TEATIME is available on GitHub (https://github.com/liliulab/TEATIME) and Zenodo (https://zenodo.org/records/17422174).