Citations

Abstract Introduction: Fibroblast growth factor receptor 2 and 3 (FGFR2 and FGFR3) are metabolic genes overexpressed in malignant epithelia. In colorectal cancer (CRC), intimate crosstalk between FGFR2/3 and epidermal growth factor receptor (EGFR) pathways has been suggested but is not fully elucidated. As a result, this study investigated associations between FGFR2/3 expression levels and clinical outcomes in patients with metastatic CRC (mCRC) enrolled in the phase III clinical trial CALGB /SWOG 80405. Methods: Data from 433 patients with mCRC enrolled in the CALGB/SWOG 80405 trial were analyzed. Patients received either bevacizumab (bev, n = 226) or cetuximab (cet, n = 207) plus chemotherapy as first-line treatment. Tumor RNA was extracted from FFPE samples and processed through the HiSeq 2500 (Ilumina) platform. Median overall survival (OS) and progression-free survival (PFS) curves were evaluated in all treatment subgroups and stratified according to high (H), medium (M), and low (L) FGFR 2 and 3 gene expression levels. Likelihood ratio tests, hazard ratios (HRs), and 95% confidence intervals (CIs) were calculated using Cox proportional hazards multivariate models, adjusting for age, sex, ECOG performance status, tumor location, number of metastatic sites, KRAS status, consensus molecular subtypes, and treatment arm. Results: Both FGFR2 and FGFR3 expression levels were associated with outcomes in patients treated with cetuximab. FGFR2-L tumors showed significantly longer OS compared to the M (HR = 1.04; 95%CI 0.71-1.51) and H (HR = 1.56; 95%CI 1.06-2.28) expression groups (35.7 vs. 31.2 vs. 23.7 months, p = 0.017). In contrast, FGFR3-L tumors were associated with shorter OS compared to the M (HR = 0.63; 95%CI 0.44-0.92) and H (HR = 0.59; 95%CI 0.41-0.85) expression groups (24.7 vs 32.4 vs 37.1 months, p = 0.0009). Significant differences in PFS were not observed. A nominally significant interaction was found between treatment and FGFR3 expression for both PFS (p = 0.02) and OS (p = 0.0003), but no interaction with FGFR2 expression. No significant associations were detected in patients treated with bev. Neither FGFR2 nor FGFR3 suggested to be prognostic markers after multivariate adjustment analyses. Conclusions: Our findings suggest a predictive role of FGFR3 in patients with mCRC undergoing anti-EGFR treatment. Differences in survival within the cet subgroup suggest that EGFR inhibitor efficacy may be modulated by FGFR2/3 signaling activity. Interestingly, FGFR2 and FGFR3 expressions were inversely and positively correlated with improved OS. The findings suggest that FGFR2 signaling minimizes the specific effects of cetuximab, while FGFR3 signaling amplifies its therapeutic effects. Further studies should determine whether these associations are seen in other anti-EGFR inhibitors and detail the mechanistic interaction between FGFR and EGFR pathways. Citation Format: Steve Soto Trujillo, Yan Yang, Michela Bartolini, Shivani Soni, Pooja Mittal, Fang-Shu Ou, Lesly Torres-Gonzalez, Unnati Hemant Shah, Jae Ho Lo, Yitzhar Goretsky, Wu Zhang, Alan Venook, Sandra Algaze, Joshua Millstein, Heinz-Josef Lenz. Differences in survival outcomes among metastatic colorectal cancer patients across fibroblast growth factor receptor 2 and 3 expression levels [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 6751.

Abstract High-grade serous ovarian cancer (HGSOC), the most prevalent and lethal ovarian cancer subtype, is typically diagnosed at an advanced stage due to the absence of effective early detection strategies. Mounting evidence indicates that HGSOC originates from the fallopian tube (FT), particularly from secretory epithelial cells in the fimbria. However, incomplete understanding of the cellular and molecular origins of HGSOC continues to hinder early interception efforts. To address this, we established a clinically and genetically annotated living FT organoid biobank at Mayo Clinic, representing over 200 patient donors. Using optimized enrichment protocols, we isolated FT epithelial stem/progenitor cells from tissue and Tao brushings, generating organoids independent of anatomical site or laterality that recapitulate native FT epithelial architecture and lineage differentiation. We applied integrated multi-omic analyses, including single-cell RNA sequencing, single-nucleus RNA/ATAC sequencing, and bulk RNA-seq, across fresh cells and organoids. Integration with public datasets produced the largest single-cell atlas of normal and high-risk FTs to date. This analysis defined high-specificity gene signatures for secretory and multiciliated lineages that outperform canonical lineage markers (e.g. PAX8, FOXJ1), identified lineage-defining transcription factors and regulatory networks, and showed concordant protein expression in the human FT epithelium. Notably, subcluster-level analysis revealed a rare hybrid epithelial-mesenchymal population transcriptionally and proteomically aligned with the mesenchymal-like subtype of HGSOC. Spatial immunoprofiling demonstrated that these cells express multiple defining markers and reside above the basement membrane, intercalated between epithelial cells and lack luminal contact, reminiscent of the histologically described peg cell population. Comparative analyses with fetal mesonephric tissues uncovered developmental parallels, and this population was stably maintained in 3D organoid cultures, providing a robust ex-vivo model to study early transformation. Together, our findings identify a rare, multipotent epithelial population with mesenchymal features as a candidate origin for mesenchymal-like HGSOC and establish the FT organoid biobank as a powerful platform for mechanistic and translational studies in ovarian carcinogenesis. Citation Format: Megan L. Ritting, Wenmei Yang, Syed Mohammed Musheer Aalam, Hui Zhao, Liang Feng, Jianning Song, Mihai G. Dumbrava, Wazim M. Ismail, Kenneth Schaufelberger, S. John Weroha, Scott H. Kaufmann, Alexandre Gaspar-Maia, Mark E. Sherman, Jamie N. Bakkum-Gamez, Nagarajan Kannan. A rare multipotent peg-like epithelial cell is a candidate cell-of-origin for high-grade serous ovarian 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 7636.

Abstract Introduction: Urine cell-free DNA (cfDNA) is a promising ultra-noninvasive analyte for genitourinary cancers (GU) cancers. In this study, we investigated urine cfDNA fragmentomic features and evaluated their ability to predict the tumor tissue of origin. Methods: A total of 204 GU cancer patients and 34 healthy adults were enrolled in this prospective study. Preoperative urine was collected from 89 bladder cancer (BC) patients (64% muscle-invasive) undergoing cystectomy, 65 renal cell carcinoma (RCC) patients undergoing nephrectomy and 50 metastatic prostate cancer (mPC) patients. Urine cfDNA was isolated and sequenced at 5x genome-wide coverage on a NovaSeq S4 flow cell. To identify malignant tissue of origin, we analyzed somatic copy number alterations (CNAs), genome-wide fragment length ratios, and 4-mer end motifs. CNAs and tumor fraction (TFx) were quantified using ichorCNA in 1-Mb windows, while chromosome arm-level fragment count z-scores and fragment length ratios were calculated in 5-Mb bins across the genome. Relative frequencies of all 256 possible 4-mer end motifs were calculated for each sample. Individual logistic regression models were developed based on each fragmentomic feature using leave-one-out cross validation (LOOCV). Outputs from these models were combined into multi-feature XGBoost models. Cohort-specific end-motif enrichments were identified using Kruskal-Wallis tests with FDR correction. Motifs were then hierarchically clustered based on their cohort-wise median frequencies to define groups of motifs with shared fragmentation patterns. Results: Across urine cfDNA in the GU cohort, BC showed the highest mean TFx at 10.0%, followed by mPC at 3.6% and RCC at 2.7%. A machine learning model based on 4-mer end motifs achieved a mean ROC AUC of 0.933 with classification accuracies of 63% for healthy, 87% for BC, 72% for mPC, and 83% for RCC. This model outperformed models based on ichorCNA (AUC = 0.767), chromosome arm fragment-count z-scores (AUC = 0.827), and fragment length ratios (AUC = 0.674). Combining end motifs with other fragmentomic features did not improve prediction accuracy. Multi-feature models incorporating end motifs with ichorCNA, chromosome arm-level z-scores, or fragment-length ratios yielded mean AUCs between 0.908-0.924, lower than the end-motif only model. Kruskal-Wallis tests identified 179 of 256 end motifs (79%) as significantly different across healthy, BC, mPC, and RCC. Hierarchical clustering of cohort-level median motifs revealed distinct end motif patterns: Motifs beginning with CT were enriched in RCC; AAAA was strongly enriched in BC; and motifs beginning with CA, AC, and AG were enriched in mPC. Conclusion: Urine cfDNA 4-mer end motifs capture GU cancer-specific biological patterns that enable accurate tissue-of-origin prediction, underscoring their promise as a diagnostic tool. Citation Format: Jessica Linford, Pradeep S. Chauhan, Irfan Alahi, Yohan Kim, Arpit Panda, Nathan Colon, Ryan Mueller, Faridi Qaium, Eric H. Kim, Melissa A. Reimers, Zachary L. Smith, Woodson W. Smelser, Fabrice Lucien-Matteoni, Aadel A. Chaudhuri. Urine cfDNA 4-mer end-motif signatures outperform other fragmentomic features for tissue-of-origin classification in genitourinary cancers [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 2590.

Abstract While missense somatic mutations in the serine/arginine rich splicing factor 2 (SRSF2) occur early in the clonal development of myeloid neoplasms, they are uniquely enriched in chronic myelomonocytic leukemia (CMML), an MDS/MPN overlap neoplasm, where their estimated prevalence is ∼50%. Short-read RNA-sequencing experiments have demonstrated that missense alterations of the proline 95 residue hotspot (SRSF2P95) globally dysregulate exon inclusion, though the full transcript structures induced by such alternative splicing events remain uncharacterized. Given the striking enrichment of SRSF2 mutations in CMML, we hypothesized that there exist unique splicing alterations contributing to the biology of this disease, and sought to profile the landscape of alternative transcription induced by SRSF2 mutations in CMML using long-read RNA-Seq. We conducted bulk long-read RNA-Seq on bone marrow mononuclear cells from 5 CMML patients with SRSF2P95H (n=3) and SRSF2P95L (n=2) mutations, with VAFs ranging 20-48%, along with 5 CMML splicing factor wildtype patients. Libraries were prepared with the Pacbio Kinnex full-length RNA kit and sequenced on a Revio SPRQ sequencer. Reads were processed and aligned using the IsoSeq pipeline, and isoform collapsing, quality control, reference comparison, and quantification were performed with IsoQuant and SQANTI3. Differential transcript and gene expression was assessed with DESeq2, while differential transcript usage was assessed with the DRIM-Seq and stageR packages. After long-read collapsing and quality control, we identified 255,505 unique transcript isoforms, of which 14,845 (∼5.8%) were novel to the reference transcriptome annotation (GENCODE v39). We identified 23 transcripts differentially expressed in SRSF2-mutant samples, including 9 novel transcripts, the majority of which (n=7) were upregulated in SRSF2-mutant cases. A higher number of transcripts (n=80) and genes (n=65) demonstrated significant changes in proportional transcript usage. Among transcripts with increased expression and usage in SRSF2-mutant cases, we identified a novel transcript of the gene YBX1, which encodes an RNA- and DNA-binding transcription factor required for cell survival and self-renewal in myeloid neoplasms. This novel transcript, which we termed YBX1ΔEx4, has an in-frame loss of exon 4, and is predicted to encode a full-length protein missing a 30 amino acid region of the cold-shock (DNA-binding) domain. Using short read RNA-Seq data from an additional 29 CMML patients, as well as an independent dataset (GSE251806) comprising 43 blast-phase CMML patients, we confirmed increased exclusion of YBX1 exon 4 in SRSF2-mutant cases, with an even greater loss of exon 4 in blast-phase SRSF2-mutant cases. We hypothesize an important role of this isoform in CMML disease progression and are currently performing functional studies to characterize the effect of YBX1ΔEx4. Citation Format: Nickolas Steinauer, Terra Lasho, Christy Finke, Pankaj Pradeep, Jenna Fernandez, Alejandro Ferrer, Moritz Binder, Abhishek Mangaonkar, Mrinal M. Patnaik, . Long-read RNA-Seq reveals a novel isoform landscape of SRSF2-mutant chronic myelomonocytic leukemia [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 1497.

Abstract Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, thus, there is an urgent need to develop more effective therapeutic options for this dismal condition. Tumor-infiltrating lymphocytes (TILs) are associated with improved response to immune checkpoint blockade in HCC, but their low abundance in most cases limits their therapeutic efficacy. Here, we demonstrate, in mice, that low-dose intratumoral immunovirotherapy with the trivalent measles, mumps, and rubella vaccine (MMR) induces superior tumor-growth delay and extended host survival compared to individually administered vaccines for measles, mumps, or rubella viruses. Further, our results show that MMR therapy synergizes with PD-1 and CTLA-4 blockade to reprogram the tumor microenvironment, resulting in increased CD8+ TIL infiltration and reduced PD-1 expression on TILs, among other effects. These changes in the immunological landscape translated into greater survival and more durable tumor-specific and memory immune responses for hosts. Comprehensive toxicology analysis revealed no evidence of MMR-induced liver or kidney toxicity after intrahepatic administration. This work reinforces an unrecognized role of MMR plus ICB in reprogramming the immune landscape in HCC through multimodal immune activation, providing a strong rationale for further development of MMR-based therapies for HCC. Citation Format: Mulu Z. Tesfay, Aleksandra Cios, Khandoker Usran Ferdous, Randal S. Shelton, Isabelle Miousse, Camila C. Simoes, Alexei Basnakian, Rangaswamy Govindarajan, Martin Cannon, Mitesh J. Borad, Bolni M Nagalo. Live attenuated MMR vaccines modulate tumor immune cell infiltration and synergize with standard of care to prolong survival in preclinical HCC models [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 4378.

Abstract Background: Colorectal cancer (CRC) remains a leading cause of cancer-associated mortality and is difficult to treat due to therapy resistance and limited durable responses. Esophageal cancer (EC) is similarly challenging, exhibiting highly aggressive and metastatic behavior that contribute to its poor prognosis. Adoptive TCR-engineered T cell (TCR-T) therapies show promise in these settings, but their efficacy is limited by suboptimal antigen specificity and an immunosuppressive tumor microenvironment (TME). To address these barriers, we isolated high-affinity TCRs from a phage-displayed TCR library targeting KRASG12V neoantigen in CRC and CTAG1B cancer/testis antigen (CTA) in EC. Methods: KRASG12V(8-16)/HLA-A11:01-restricted and CTAG1B(157-165)/HLA-A02:01-restricted TCRs were isolated from an optimized phage-displayed TCR library generated from healthy donor CD8+ T cells through high-throughput biopanning. Binders were screened against antigen-family peptides and irrelevant controls by phage ELISA to eliminate cross-reactivity. Binding affinities were measured by SPR. Antigen-specific TCRs (asTCRs) were cloned into lentiviral vectors and transduced into primary human T cells. TCR-T function was assessed using peptide-pulsed T2 cells and KRASG12V+ or CTAG1B+ tumor cells through LDH-release cytotoxicity and cytokine secretion assays. Normal human cells were used to evaluate off-target toxicity. In vivo antitumor activity was tested in NOD/SCID-IL2rγ-/- xenografts with histological confirmation of TCR-T infiltration. Results: Next-generation sequencing confirmed that the 109-scale library contained >90% functional genes and >80% sequence diversity. Three high-affinity TCRs were identified for each antigen (KD as low as 1.07 μM). These TCRs exhibited strict antigen specificity with no detectable cross-reactivity. TCR-T cells expressing asTCRs showed potent antigen-dependent cytotoxicity against KRASG12V+/HLA-A11:01+ colorectal and CTAG1B+/HLA-A02:01+ esophageal tumor cells (up to 80% lysis, p<0.01), accompanied by robust IFN-γ and TNF-α secretion (>1000 pg/mL, p<0.01). No killing was observed against any of seven healthy cell types. TCR-T cells remained effective at low effector-to-target ratios and maintained stable proliferation and effector-memory phenotypes. In xenograft models, KRAS- and CTAG1B-targeted TCR-T cells induced significant tumor regression compared to unmodified T cell controls (p<0.05), with increased effector TCR-T infiltration, and reduced antigen escape. Conclusions: This work establishes a rapid and robust platform for isolating potent, safe, antigen-specific TCRs targeting both oncogenic driver mutation (KRASG12V) and an immunogenic CTA (CTAG1B). TCR-T cells demonstrated strong antitumor activity in vitro and in vivo, supporting the translational potential of this approach for solid tumor treatment. Citation Format: Obed Boadi Amissah, Wenfang Chen, Zhiyuan Li, Gloria Bora Kim. KRASG12V- and CTAG1B-targeted TCR-T cells from phage TCR library suppress colorectal and esophageal tumors [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 5618.

Abstract Gastrointestinal (GI) cancers are rising in people < 50 and remain major causes of cancer mortality despite therapeutic advances. Chimeric antigen receptor T (CART) cell therapy has transformed treatment of hematological cancers. However, its efficacy in GI cancers has been hindered by antigen heterogeneity and immunosuppressive cancer associated fibroblasts (CAFs) in the tumor microenvironment (TME.) Here we aimed to 1) study CART-CAF interactions and 2) engineer CART cells capable of eliminating CAFs to overcome their inhibitory role and enhance their antitumor activity in GI cancers. We first analyzed primary CAFs from 20 GI cancer patients. CAFs showed high fibroblast activation protein (FAP) expression and significantly suppressed T cells proliferation (26-77%, p≤0.0001). We next generated CART cells capable of safely eliminating both GI cancer cells and CAFs. To target cancer cells, we developed a novel nanobody (VHH)-based CAR against Claudin18.2, a GI cancer specific target. To avoid off-tumor toxicity in healthy FAP+ tissues, we incorporated an inducible FAP-CAR using the synNotch platform, enabling conditional targeting upon engagement with Claudin18.2+ cells, restricting FAP targeting within the TME. VHH Claudin18.2 CART cells showed superior specificity and efficacy vs scFv-based CART cells (hu8E85 or zolbetuximab). In Claudin18.2+ SNU-C2B xenografts, tumors were 92mm3 (VHH) vs 48mm3 (huE85) vs 211mm3 (zolbetuximab) on day 23 post-CART, median survival undefined vs 17 days vs 23 days respectively; p=0005. In Claudin18.2+ LS411N xenografts, tumors were 315mm3 vs 106mm3 vs 382 mm3 respectively at 23 days post-CART, median survival undefined vs 19 days vs undefined respectively; p=0004. VHH Claudin18.2 CART cells showed no off-tumor effects, while hu8E85 CART induced significant toxicity. We next established a co-engraftment model. NSG mice received 1x106 SNU-C2B and 6x106 CAFs from a GI cancer patient. At 500 mm3 tumors, mice received 5x106 VHH Claudin18.2 or inducible Claudin18.2/FAP CART. Dual-targeting CART cells showed greater antitumor response (728mm3 vs 320mm3, p=0.0011 at day 20 post-CART), expansion (61 vs 2,350 CART/µl of blood, p=0.0037), and tumor infiltration (8.71 x 105 vs 1.03 x 106 CART/g of tumor, p<0.0001). This was also observed in gastric, colon, and pancreatic PDX models, with no observable off-tumor cytotoxic effects with inducible dual-targeting Claudin18.2/FAP CART. Our findings demonstrate that our novel VHH-based Claudin18.2 CART cells are highly specific and effective against GI malignancies, and that inducible Claudin18.2/FAP dual-targeting CART cells safely remodel the TME into an immune-permissive state. This approach represents a mechanistically innovative and translationally relevant strategy to overcome CAF-mediated immunosuppression, enhance CART cell efficacy, and improve clinical outcomes in refractory solid tumors. Citation Format: Omar L. Gutierrez Ruiz, Jennifer Feigin, Brooke Kimball, R. Leo Sakemura, Elizabeth L. Siegler, Dominic A. Skeele, Ateka Saleh, Claudia Manriquez Roman, Ismail Can, Olivia Sirpilla, Kun Yun, Carli M. Stewart, Mehrdad Hefazi, Yoh Yamaguchi, Lionel Aurelien Kankeu Fonkoua, Long K. Mai, Truc Huynh, Sophia Y. Goldberg, Grace E. DeFranco, Sara Foote, Ekene Johnkennedy Newton Ogbodo, Hong Xia, Michael Redig, Matthew L. Matthew, Tamiel Nichole Turley, Skyeler Klinge, John Alton Copland, Saad J. Kenderian. Optimized CART cell therapy to remodel the tumor microenvironment and eliminate GI malignancies [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 1525.

Abstract The sentinel lymph node (SLN) is often the first site of melanoma spread and a critical immune checkpoint that determines local tumor dissemination to systemic metastasis. Although lymph nodes are central to antitumor immunity, the mechanisms that render them permissive to metastasis remain poorly understood. This study used spatial imaging to characterize immune and stromal phenotypic alteration in tumor-negative SLNs (SLN-) and tumor-positive SLNs (SLN+) from patients with cutaneous melanoma, compared to non-melanoma-associated normal lymph nodes (NLN). We performed multiplex immunofluorescence (MxIF) analysis of formalin-fixed, paraffin-embedded lymph node tissues from patients with cutaneous melanoma and control subjects, including two subjects per group (NLN, SLN-, and SLN+). Furthermore, quantitative single-cell segmentation and classification using QuPath generated median fluorescence intensities for immune, stromal, and tumor markers across LN, SLN- and SLN+ regions of interest. In addition to the initial analysis, we employed spatial neighborhood analysis to characterize markers expression profiles surrounding target immune population. This approach quantified local microenvironment based on marker normalized intensity-based co-expression pattern and stromal remodeling signatures. We grouped these markers based on the functional panels representing T-cell activation and exhaustion, myeloid polarization, tumor interaction, stromal remodeling, and apoptosis pathways providing insights into how prometastatic niche evolve within SLN. Normalized data, via centered log ratio (CLR) transformation and Gaussian Mixture Model (GMM) gating was analyzed using principal component analysis, UMAP/tSNE embedding, and hierarchical clustering in in python. Progressive immune remodeling was observed from normal NLN to SLN+, including expansion of regulatory T cells, enrichment of exhausted T cells as well as myeloid-derived suppressor cells (MDSC), and transition toward an M2-like macrophage phenotype. The spatial data from SLN- samples showed an early sign of immune suppression, with reduced cytotoxic capacity. These results show that SLN gets features of peripheral immune tolerance for melanoma early, prior to pathological dissection of metastatic spread supporting the existence of a pre-metastatic niche that facilitates lymphatic colonization of tumor cells. This work provides insights into early immune escape and may inform strategies for immunotherapy and early intervention in melanoma. Citation Format: Shankar Suman, Wendy K. Nevala, Noah A. Stueven, Raymond M. Moore, Chathu L. Atherton, Jeffrey E. Johnson, Ray Guo, James W. Jakub, Svetomir N. Markovic. Spatial remodeling of immune and stromal microenvironments in sentinel lymph nodes during melanoma progression [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 6193.

Abstract Background: PD-L1 is best known for suppressing antitumor immunity at the cell surface, yet emerging evidence suggests additional nuclear functions. Previous studies from our laboratory demonstrated that nuclear PD-L1 interacts with the cohesin complex to maintain chromosomal stability. However, the upstream regulatory mechanism governing this interaction and its immunological consequences remain unknown. Methods and Results: Using acetylation-site prediction, mutagenesis, and structural modeling, we identified lysine 178 (human) / 177 (mouse) as a conserved acetylation switch regulating PD-L1 function. Deacetylation of PD-L1 (K178R/K177R) disrupted PD-L1-PDS5B/cohesin binding without affecting subcellular localization. This led to defective sister chromatid cohesion, micronuclei formation, and cytosolic DNA accumulation. RNA-seq, qPCR, and immunoblotting demonstrated robust activation of the cGAS-STING-IFN-γ signaling axis and enhanced MHC-I antigen-presentation machinery in PD-L1 K178R cells. Functionally, K178R cells proliferated more slowly and were highly susceptible to PBMC-mediated killing in co-culture assays, with further enhancement upon anti-PD-L1 antibody treatment. In xenograft models (NSG mice), tumors expressing PD-L1 K178R grew significantly slower than WT. In immunocompetent C57BL/6 syngeneic models, PD-L1 K177R tumors showed reduced growth and heightened responsiveness to PD-L1 blockade. Structural and biochemical analyses confirmed that PD-L1 acetylation at K178/K177 stabilizes its interaction with the cohesin complex, thereby preventing chromosomal instability-induced immune activation. Conclusions: We identify a conserved PD-L1 acetylation switch that couples cohesin stability to innate immune activation. PD-L1 deacetylation promotes chromosomal instability, activates cGAS-STING-IFN-γ signaling, enhances antigen presentation, and sensitizes tumors to PD-L1 immunotherapy. These findings uncover a previously unrecognized regulatory axis linking tumor genomic integrity to immune recognition, providing a mechanistic foundation for targeting PD-L1 acetylation in cancer treatment. Citation Format: Meijie Wang, Jia Yu, Liewei Wang. A pd-l1 acetylation switch coordinates chromosome cohesion and antitumor immunity [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 7391.

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and remains difficult to treat due to the absence of targeted therapies. While recent advances in immunotherapy, particularly with immune checkpoint inhibitors (ICIs), have transformed the treatment landscape for many cancers, response rates to ICIs remain low for TNBC, largely due to tumor-intrinsic immune evasion mechanisms. Objective: We identify and characterize a novel immune evasion pathway in TNBC driven by deregulation of cancer cell-intrinsic protease-activated receptor 1 (PAR1), a thrombin receptor frequently upregulated in aggressive tumors. Methods and Results: Through integrative bioinformatic analyses, genetic perturbations, xenograft models (immunodeficient and immunocompetent), flow cytometry-based tumor immune profiling, and RNA-seq, we demonstrate that PAR1 activation enhances immune evasion in TNBC. Mechanistically, we show that the paracaspase MALT1 acts as a critical downstream effector of PAR1 signaling and promotes immune escape by driving PD-L1 expression in TNBC cells. Functional assays reveal that genetic depletion of MALT1 or PD-L1 increases TNBC cell susceptibility to T cell-mediated cytotoxicity in vitro and significantly suppresses tumor growth in vivo. Notably, through knock-in and rescue experiments using a catalytically inactive MALT1 mutant, we reveal that MALT1’s scaffolding - rather than protease - function is essential for PD-L1 regulation. Immune cell depletion assays further identify CD4+ T cells, CD8+ T cells, and natural killer (NK) cells as key mediators of the anti-tumor immune response suppressed by the PAR1-MALT1 axis. These findings are supported by human TNBC specimen analyses, where MALT1 expression inversely correlates with T cell activation and positively associates with PD-L1 in PAR1-high, but not PAR1-low, tumors. Conclusion: Our study reveals immune evasion as a novel mechanism of PAR1-driven breast cancer pathogenesis, mediated by a PAR1/MALT1/PD-L1 signaling cascade. These findings bridge a critical gap by positioning MALT1 at the interface between tumor-intrinsic signaling and the immune microenvironment - two areas of CARMA-BCL10-MALT1 (CBM) complex research that have largely evolved in parallel over the years. Importantly, targeting MALT1 may enhance immunotherapy efficacy in TNBC. Given the availability of MALT1 inhibitors in clinical development, our work supports combining MALT1 inhibition with ICIs or other immune-oncology agents as a promising strategy to overcome immune resistance in TNBC. Citation Format: Dong Hu, Prasanna Ekambaram, Zheqi Li, Linda Klei, Maria L. Beecher, Yi Liu, Zongyou Cai, John Little, Jeffrey A. Meridew, Jia-Ying Lee, E. Aubrey Thompson, Tullia C. Bruno, Lidija Covic, Seung-Oe Lim, Anushka Dongre, Heide L. Ford, Mien-Chie Hung, Adrian V. Lee, Steffi Oesterreich, Linda McAllister-Lucas, Peter C. Lucas. A cancer cell-intrinsic PAR1/MALT1/PD-L1 signaling pathway drives immune evasion in triple-negative breast 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 7392.