Supplementary Figure 5. TCR-T cells lack recognition of normal tissue cell lines.

Supplementary Figure 2. FOXM1 epitopes are eluted on H1975.

Abstract FOXM1 is highly expressed in various cancer types and considered a key driver of cancer progression. Accordingly, we evaluated the immunogenicity of FOXM1 and investigated the feasibility of targeting this transcription factor using T-cell receptor (TCR) engineering. We identified epitopes derived from FOXM1 which were immunogenic on HLA-A*02:01, HLA-A*24:02, and HLA-A*23:01, endogenously processed and presented, and resulted in T-cell activation and cytotoxic T-cell responses. Following the generation of TCR-T cells, sensitivity and specificity were confirmed by peptide dose–response and X-scan, respectively. Most importantly, adoptive transfer of TCR-engineered T cells led to a significant reduction in tumor growth, as well as significantly prolonged survival in a tumor-bearing immunocompromised murine model. Our studies confirm the immunogenicity of FOXM1 and feasibility of targeting this antigen using TCR engineering.

Supplementary Figure 7. FOXM1-specific TCR-T do not lead to toxicity in vivo.

FOXM1 is highly expressed in various cancer types and considered a key driver of cancer progression. Accordingly, we evaluated the immunogenicity of FOXM1 and investigated the feasibility of targeting this transcription factor using T-cell receptor (TCR) engineering. We identified epitopes derived from FOXM1 which were immunogenic on HLA-A*02:01, HLA-A*24:02, and HLA-A*23:01, endogenously processed and presented, and resulted in T-cell activation and cytotoxic T-cell responses. Following the generation of TCR-T cells, sensitivity and specificity were confirmed by peptide dose-response and X-scan, respectively. Most importantly, adoptive transfer of TCR-engineered T cells led to a significant reduction in tumor growth, as well as significantly prolonged survival in a tumor-bearing immunocompromised murine model. Our studies confirm the immunogenicity of FOXM1 and feasibility of targeting this antigen using TCR engineering.

HER2 mutations are oncogenic drivers in 1 to 6% of non small cell lung cancers (NSCLC), but therapeutic resistance limits the durability of current HER2 targeted treatments. Here, we identify T cell receptors (TCRs) targeting recurrent HER2 hotspot mutations as a potential immunotherapeutic strategy for HER2 mutant NSCLC. Using neoepitope prediction and antigen specific T cell enrichment, we isolated HLA A*02:01 restricted TCRs recognizing HER2 A775insYVMA, S310F, and G776delinsVC mutations, collectively covering approximately 60% of HER2 mutant NSCLC. These TCRs selectively recognized mutant HER2 epitopes without detectable wild type reactivity and some displayed cross recognition of related hotspot variants, expanding the spectrum of targetable tumors. The G776delinsVC specific TCR also exhibited coreceptor independent activity showcased by its ability to activate CD4+ T cells. Importantly, time lapse single cell flow cytometry analyses demonstrated that TCR engineered T cells repeatedly reacquired activated polyfunctional states following serial antigen stimulation, while serial tumor rechallenge assays confirmed sustained cytotoxic activity across multiple rounds of tumor killing. These findings identify recurrent HER2 mutations as shared immunotherapeutic targets and provide a foundation for the development of TCR based therapies for HER2 mutant NSCLC.

MOTIVATION:Examination of T cell receptor (TCR) clonality has become a way of understanding immunologic response to cancer and its interventions in recent years. An aspect of these analyses is determining which receptors expand or contract statistically significantly as a function of an exogenous perturbation such as therapeutic intervention. RESULTS:We characterize the commonly used Fisher's exact test approach for such analyses and propose an alternative formulation that does not necessitate pairwise, within-patient comparisons. We develop this flexible Bayesian longitudinal mixture model that accommodates variable length patient followup and handles missingness where present, not omitting data in estimation because of structural practicalities. Once clones are partitioned by the model into dynamic (expanding or contracting) and static categories, one can associate their counts or other characteristics with disease state, interventions, baseline biomarkers, and patient prognosis. We apply these developments to a cohort of prostate cancer patients who underwent randomized metastasis-directed therapy or not. Our analyses reveal a significant increase in clonal expansions among metastasis-directed therapy (MDT) patients and their association with later progressions both independent and within strata of MDT. Analysis of receptor motifs and VJ gene enrichment combinations using a high-dimensional penalized log-linear model we develop also suggests distinct biological characteristics of expanding clones, with and without inducement by MDT. AVAILABILITY AND IMPLEMENTATION:An example model implementation in R/STAN is available at doi.org/10.5281/zenodo.21209546. SUPPLEMENTARY INFORMATION:Supplementary material includes simulation results, longitudinal mixture model component derivation, HLA typing analysis, and stability selection for the VJ gene family.

Abstract Background: Loss of tumor-specific MHC class I (tsMHC-I) is a recurrent feature of lung adenocarcinoma (LUAD) progression and is associated with immune escape and poor outcomes. However, how tsMHC-I downregulation shapes tumor evolution and remodels the immune microenvironment remains unclear. Methods: We integrated single-cell RNA sequencing, spatial transcriptomics, and multiplex imaging from human LUAD samples spanning precancerous lesions to advanced tumors, together with longitudinal genetically engineered mouse models (GEMMs). Functional assays—including neutrophil co-culture, assessment of neutrophil extracellular traps (NETs), and in vivo tumor studies—were performed to define mechanistic consequences of tsMHC-I loss. Results: Progressive tsMHC-I downregulation was observed across LUAD evolution and validated in GEMMs. Single-cell trajectory analysis showed that reduced tsMHC-I is linked to loss of alveolar type II identity, increased chromosomal instability, heightened tumor plasticity, and acquisition of epithelial-mesenchymal transition (EMT) programs. Functionally, tsMHC-ILow malignant cells displayed increased invasiveness and metastatic capacity. Spatial and immunophenotypic profiling revealed that tsMHC-I downregulation reshapes the tumor microenvironment toward a neutrophil-enriched, cytotoxic cell-excluded niche. tsMHC-ILow regions exhibited dense tumor-associated neutrophil aggregates and depletion of CD8+ T cells, NK cells, and other effector populations. Mechanistic studies showed that tsMHC-ILow malignant cells induce robust NET formation. In vitro assays identified Annexin A2, a tumor-secreted protein, as a key driver of NET induction. Annexin A2 expression and secretion were consistently elevated in tsMHC-ILow cells across human and mouse models, and Anxa2 knockdown markedly reduced NET formation. Targeting NETs in vivo with DNase I selectively suppressed growth and metastatic spread of tsMHC-ILow tumors, restored cytotoxic immune infiltration, and reversed the immune-excluded phenotype. In tsMHC-I heterogeneous tumors, combining NET inhibition with anti-PD-1 enhanced tumor control and survival. Conclusions: Our integrated analyses reveal that tsMHC-I downregulation drives neutrophil recruitment and NET formation through Annexin A2 secretion, creating an immunosuppressive barrier that promotes LUAD progression and limits immunotherapy response. These findings identify a previously unrecognized tsMHC-I-NET axis as a therapeutic vulnerability in LUAD. Citation Format: Yanhua Tian, Jian-Rong Li, Bo Zhu, Jared Fradette, Hong Chen, Zhubo Wei, Jie Ye, Shao-Wei Lu, Andrew Y. Liu, Samrat T. Kundu, Haoyi Wu, Shucheng Miao, Xiuning Le, Linghua Wang, Jia Wu, Alexandre Reuben, John V. Heymach, Andy Futreal, Honami Naora, Chao Cheng, Don L. Gibbons, Jianjun Zhang. Spatiotemporal analysis reveals tsMHC-I downregulation-induced neutrophil extracellular traps as a driver of lung adenocarcinoma neoplastic evolution [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 200.

Supplementary Table 1. Predicted peptides with corresponding HLA alleles.

Abstract Loss-of-function mutations in RBM10, encoding a protein involved in the regulation of alternative splicing, are observed in ∼10% of non-squamous NSCLC and are enriched in tumors harboring activating mutations in KRAS, yet little is known about how RBM10 inactivation promotes lung cancer progression or influences response to standard-of-care systemic therapies. Here, we elucidated the cooperative interplay between oncogenic KRAS activation and RBM10 loss in NSCLC pathogenesis using a novel genetically engineered mouse model with conditional RBM10 deletion, as well as multiple isogenic syngeneic allograft models that faithfully recapitulate RBM10-deficient human lung adenocarcinoma. We found that loss of RBM10 accelerates KRAS-mutant NSCLC progression by fostering the establishment of a tolerogenic, myeloid cell-rich tumor immune microenvironment (TIME). Mechanistically, RBM10 loss promoted R-loop accumulation and chronic DNA damage signaling that engaged the non-canonical TRAF6-STING pathway in a cGAS-independent manner, leading to sustained NF-κB activation. We identified several cytokines and chemokines, canonical targets of NF-κB signaling, such as IL-1β, IL-6, TNFα, and MCP-1, that were upregulated in RBM10-deficient cells. This NF-κB-driven secretome promoted the development of an inflamed, TIME characterized by accumulation of suppressive myeloid cell subsets - most notably monocytes and M2-like macrophages, and dysfunctional tumor infiltrating lymphocytes (TILs) thereby fostering immune evasion and cancer progression. Furthermore, we exploited the RNA-seq database of human lung adenocarcinoma from The Cancer Genome Atlas (TCGA) and again found that RBM10 loss was significantly linked to impaired DNA damage response, upregulation of the HALLMARK_TNFA_SIGNALING_VIA_NF-κB, and enrichment of M2-macrophages. Strikingly, targeting the CSF1/CSF1R axis with an anti-CSF1R antibody significantly curtailed the growth of RBM10-deficient tumors in syngeneic immunocompetent models and synergized with anti-PD-1 therapy to promote tumor regression. In conclusion, our findings uncovered a novel critical role of RBM10 inactivation in driving immune escape and PD-1 inhibitor resistance in KRAS-mutant NSCLC and suggest a potential therapeutic strategy by co-targeting suppressive myeloid cells to improve cancer immunotherapy for patients bearing KRAS;RBM10 co-mutated tumors. Citation Format: Minh Truong Do, Teng Zhou, Mhd Yousuf Yassouf, Richard Lee, Obada E. Ababneh, Yanhua Tian, Leticia B. Rodriguez, Jayanthi Gudikote, Haniel A. Araujo, Stephanie T. Schmidt, Jing Wang, Frank R. Rojas Alvarez, Luisa M. Solis Soto, Marcelo V. Negrao, Alexandre Reuben, Don L. Gibbons, Jianjun Zhang, John V. Heymach, Ferdinandos Skoulidis. RBM10 loss promotes KRAS-mutant non-small cell lung cancer immune tolerance and PD-1 inhibitor resistance via a non-canonical STING/ NF-κB axis [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 6989.

Supplementary Figure 6. FOXM1-restricted TCR-T cells are specific.

PURPOSE We tested the hypothesis that adding metastasis-directed therapy (MDT) to standard-of-care (SOC) systemic therapy improves progression-free survival (PFS) among patients with oligometastatic disease. METHODS EXTEND was a multicenter randomized phase II trial. Patients with 1-5 metastases were randomly assigned to MDT + SOC versus SOC in one of the six baskets (breast, pancreas, kidney, two prostate baskets, and an other basket) with basket-specific stratification and powering. PFS, the primary end point, was prespecified in the per-protocol set within each basket, across all baskets, and across all baskets excluding the prostate baskets. Exploratory end points included circulating tumor DNA (ctDNA) and immune profiling. RESULTS From 2018 through 2023, 521 patients were screened, 350 were randomly assigned, and 334 were analyzed per protocol (MDT + SOC, n = 166; SOC, n = 168). Radiotherapy was used as MDT for 98% of metastases (370/379). Overall, after a median follow-up of 53 months, PFS was improved with MDT + SOC (hazard ratio [HR], 0.54 [95% CI, 0.41 to 0.72], P < .001). Similarly, PFS was improved when excluding the prostate baskets (HR, 0.60 [95% CI, 0.40 to 0.89]). Within each basket, PFS superiority was identified for the pancreas, prostate, and other baskets, whereas the breast and kidney baskets were inconclusive. At enrollment, detectable ctDNA correlated with shorter PFS and survival; by contrast, ctDNA clearance 3 months postenrollment correlated with improved survival. MDT + SOC-induced systemic immune activation was most pronounced among baskets demonstrating PFS superiority. CONCLUSION The phase II EXTEND trial supports the addition of MDT to SOC for oligometastatic disease. Histology-specific efficacy signals were identified for phase III testing. Translational insights suggest the potential for optimizing the definition of oligometastasis using ctDNA and point to systemic immune responses as a possible mechanism of benefit from MDT.

Oncogenic KRAS mutations drive a substantial proportion of lung cancers and are linked to poor prognosis, positioning KRAS as a compelling target for cellular immunotherapy. Thirty-five percent of lung adenocarcinomas harbor the KRAS G12C, G12V, G12D mutations. Here, we developed HLA-A*03:01- and HLA-A*11:01-restricted T cell receptors (TCR) targeting the most prevalent G12C and G12V KRAS hotspot mutations in lung adenocarcinoma. Predicted high affinity peptides were screened using our TCR discovery and validation pipeline, and functional assessment was performed to determine sensitivity, specificity, and cytotoxic potential of TCR-engineered T cells. We discovered and validated 5 novel TCRs targeting KRAS G12C and G12V 9-mers, each of which demonstrated an ability to recognize and lyse tumor cells endogenously presenting mutant KRAS on HLA-A*03:01 or HLA-A*11:01. Notably, several TCRs demonstrated distinct modes of cross-reactivity, including peptide degeneracy across KRAS G12 variants, HLA degeneracy across HLA-A*03:01 and HLA-A*11:01, or dual degeneracy across both KRAS and HLA, thereby broadening the treatable target populations. TCRs that recognize the KRAS hotspot shared sequence motifs were found in several lung cancer patients. Our study highlights the successful generation of multi-valent KRAS-specific TCRs and supports the feasibility of targeting shared KRAS neoantigens through TCR engineering in lung cancer.

Supplementary Figure 1. FOXM1 is minimally expressed in healthy organs.

T cells have important functions in development and disease processes through T cell receptor (TCR)-dependent activities. Many tools were developed to predict the binding between TCRs and antigens. However, one of the uncertainties is whether such tools can decipher how small changes in the TCRs or antigenic peptides contribute to binding. We develop a deep learning model, pMTnet-omni, which not only predicts the binding vs. non-binding of TCRs towards pMHCs, but also distinguishes the stronger vs. weaker binding of TCRs similar in sequence. We leverage this capability to interpret the biological rules that govern TCR-antigen pairing. This also enables pMTnet-omni to accurately predict variant TCRs with desired stronger or weaker binding to the antigen, in conjunction with a Lab-in-the-Loop (LiL) mechanism. We show that pMTnet-omni can also predict binding of TCRs towards similar pMHCs. Overall, we provide a flexible toolkit for research and translational applications involving antigens and TCRs.

Supplementary Figure 9. FOXM1 TCR-T cells are detectable in the periphery at time of sacrifice.

Abstract Lynch Syndrome (LS) provides the perfect context to understand DNA mismatch repair deficient carcinogenesis, which is characterized by neoplastic lesions with high rates of shared neoantigens eliciting adaptive immunity through T cell receptor (TCR) recognition. However, the TCR landscape in LS carriers remains unexplored. Here, we perform TCR sequencing of 277 blood samples from LS cancer survivors, previvors, and controls, as well as matching colorectal cancers and pre-cancers. We show that up to 41% of the most expanded TCRβs from colorectal neoplasms are detectable in the blood of LS carriers, while showing minimal expansion in controls. In addition, we develop and validate a classification model that distinguishes LS carriers from controls using circulating TCRβs signatures associated with LS independent of thecancer history and with cancer-free LS previvors. Together, our findings characterize circulating and tissue TCRβs associated with LS, thus representing a step toward identifying blood-based TCR biomarkers for immune surveillance.

Tumor evolution involves genetic, transcriptional, and phenotypic alterations that shape cancer cell behavior and interactions with the microenvironment. While single-cell technologies have advanced our understanding of this process, spatial dynamics remain incompletely characterized. Here, whole-exome sequencing (WES), imaging mass cytometry (IMC), and spatial transcriptomics (ST) were integrated to study molecular evolution and immune responses in two lung adenocarcinoma (LUAD) mouse models: a genetically engineered model (129S4/Sv-Kras LSL-G12D , termed 129S4 K) and a carcinogen-induced precancer model (129S4 U). Compared to 129S4 K, 129S4 U tumors exhibited higher mutational, neoantigen but lower copy number variation (CNV) burdens at matched developmental timepoints, consistent with findings of higher mutational burden in human smoking-related LUAD than nonsmoking LUAD. We profiled over 1.4 million spatial single cells from 284 IMC regions of interest and 51,531 spatial transcriptomic spots from 156 lesions across 141 mice. Macrophage abundance increased with tumor progression, while CD8 T-cell and B-cell densities declined in late-stage LUAD. 129S4 U showed greater immune infiltration in both tumor and adjacent normal tissue, higher T-cell cytotoxicity signature score in line with its higher mutational and neoantigen burdens. LUAD progression was marked by early morphological shifts and late-stage changes in cell states and interactions. These data define spatial and genetic landscapes of LUAD development and provide a framework for investigating immune evolution and therapeutic strategies in early carcinogenesis.