Abstract Introduction: Chronic lymphocytic leukaemia (CLL) cells abnormally express lipoprotein lipase (LPL), an enzyme typically restricted to adipocytes and myocytes for lipid-mediated energy utilisation. This enables CLL cells to store and utilise lipids, potentially competing with or diverting resources from healthy tissues. In vitro studies suggest that reducing fatty acid availability may limit CLL proliferation; however, little is known about how patients can modulate this process in vivo. Exercise training offers a systemic, non-pharmacological approach to counter metabolic dysregulation, with potential benefits for tumour control and overall health. Methods: We conducted a 12-week exercise trial involving five treatment-naive (TN-CLL) and five previously treated (Td-CLL) patients. We assessed the metabolic fate of ingested lipids before (Baseline) and after (Post-Intervention) the program. Patients consumed a meal containing 200mg palmitic acid tracer (13CPA), and blood samples were collected hourly for 3 hours (T0h-T3h). We assessed 13CPA enrichment in plasma triacylglycerol (TAG) and non-esterified fatty acids (NEFA), and total fatty acids in immune cells (PBMC) using mass spectrometry, and data were analysed using RM-ANOVA. Results: Post-meal ingestion, 13CPA enrichment in plasma TAG and NEFA increased steadily from T1h-T3h (p<0.001). At Baseline T3h, TN-CLL exhibited higher plasma 13CPA-TAG and unlabelled PA-TAG incorporation than Td-CLL (p<0.001). Post-Intervention T3h, TN-CLL 13CPA-TAG levels decreased (p<0.05) and were no longer significantly different than Td-CLL. TN-CLL 13CPA-NEFA enrichment increased post-Intervention compared to Td-CLL (p<0.05), suggesting enhanced 13CPA-TAG hydrolysis. Similarly, 13CPA uptake into PBMCs, which was higher in TN-CLL at Baseline T3h (p<0.05), reduced Post-intervention. Conclusion: This pilot study demonstrates the feasibility of stable isotope tracing to assess in vivo lipid uptake in CLL. Exercise training in TN-CLL patients reduced lipid uptake, suggesting a shift towards a more balanced and healthier metabolic profile. Further research is needed to determine whether exercise can disrupt the lipid dependence of CLL cells. Citation Format: Uzma Zaheer, Ellie Miles, Angela Avramovska, Vithushan Srikumaran, Andrew Hulton, Long Li, Caitlin Jeary, Andrea Sitlinger, Renata Walewska, Barbara Fielding, David Bartlett. Rebalancing systemic and cellular energy dysmetabolism in Chronic Lymphocytic Leukemia through exercise training [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 3269.

Abstract Background: ABI1 (Abelson interactor-1) is classically recognized as a multifunctional adaptor protein with homeostatic roles in cancer biology. It functions as a tumor suppressor in some cancer such as prostate cancer, yet exhibits oncogenic activity in other cancers such as for example breast cancer. Historically, ABI1 has been studied for its actin-cytoskeleton-associated functions—including cell-cell adhesion, cell motility, and lamellipodia formation—as well as its role in regulating major signaling hubs such as c-Abl, PI3K, and Src. Our recent findings reveal an unanticipated function of ABI1: direct DNA binding mediated by a conserved homeodomain homology region (HHR). This discovery led us to hypothesize that ABI1 may act as a previously unrecognized transcriptional regulator. Here, we sought to define the molecular mechanisms through which ABI1 contributes to transcriptional control. Methods: To determine sequence specificity and genomic occupancy, we performed ChIP using HHR-intact and HHR-mutant ABI1 constructs, complemented by in vitro DNA binding assays using purified proteins. Subcellular fractionation and chromatin enrichment assays assessed ABI1 nuclear localization and association with chromatin. ABI1-interacting transcriptional machinery was identified through co-immunoprecipitation (co-IP). RNA-seq comparing cells expressing wild-type ABI1 versus an HHR-defective DNA-binding mutant defined ABI1-dependent transcriptional outputs. Results: ABI1 binds DNA both in vitro and in vivo and displays reproducible sequence motifs from integrated ChIP and in vitro binding analyses. ABI1 variants containing an intact HHR domain localize preferentially to the nucleus and chromatin fractions. Co-IP studies identify ABI1 as a component of a defined transcriptional complex. RNA-seq analyses reveal that HHR-mediated DNA binding is required for a discrete ABI1-dependent transcriptional program. Conclusions: We identify ABI1 as a novel DNA-binding protein with sequence preference and transcriptional regulatory capacity mediated through its HHR domain. These findings expand the functional repertoire of ABI1 beyond actin regulation and kinase signaling, providing the first mechanistic framework for ABI1-driven transcriptional control. Citation Format: Kate Livingston, XIANG Li, Kevin M. Lin, Leszek Kotula. Mechanistic dissection of ABI1 as DNA-binding transcriptional regulator in cancer cells [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 7245.

Abstract Perineural invasion (PNI)—the infiltration of tumor cells within, around, or through nerve fibers—is a hallmark of aggressive tumor biology and an independent predictor of poor prognosis in head and neck squamous cell carcinoma (HNSCC). Despite its high prevalence, reported in up to 90% of cases, the molecular and spatial mechanisms enabling PNI remain poorly characterized. Here, we apply spatial transcriptomics to delineate the cellular, molecular, and microenvironmental features that drive PNI and its associated immune contexture. Spatial transcriptomic datasets (GSE300147, GSE252265, and GSE281978) were processed using standardized pipelines for quality control, normalization, and unsupervised clustering (UMAP). Cell populations were annotated based on canonical markers, identifying distinct epithelial, neural, Schwann, immune, and axon guidance-related clusters. Composite module scores were calculated to quantify PNI activity, neuronal and Schwann cell identity, axon guidance signaling, and immune infiltration. Correlation analyses and Gene Ontology (GO) enrichment were used to define key biological pathways associated with spatially resolved PNI phenotypes. UMAP visualization revealed spatially distinct neural-enriched tumor clusters co-localizing with Schwann and axon guidance signatures, indicating a neural-like invasive niche. Correlation analysis demonstrated strong associations between PNI and neuronal (r = 0.89) and axon guidance (r = 0.65) programs, with moderate correlation to Schwann cell activity (r = 0.49). In contrast, PNI exhibited weak negative associations with T-cell (r = -0.26) and macrophage (r = -0.12) scores, consistent with an immune-excluded perineural microenvironment. GO enrichment further supported these trends. PNI-high regions showed activation of pathways involved in chemokine-mediated signaling, cellular adhesion, and fluid and lipid transport—processes that may facilitate neural infiltration and axon-tumor communication. PNI-low regions, by contrast, were enriched for immune-related functions including T-cell and B-cell receptor signaling, interleukin-2 production, and tumor necrosis factor regulation, consistent with active antitumor immunity. Collectively, these data reveal that PNI progression coincides with the emergence of a neural-dominant, immune-silent niche enriched for chemotactic and axon guidance signals. Together, these findings define the spatial neuroimmune landscape of PNI in HNSCC, implicating reciprocal nerve-tumor signaling and localized immune evasion as synergistic drivers of invasion. By integrating spatial resolution with transcriptomic profiling, this study provides mechanistic insight into how cancer cells remodel the perineural microenvironment to support invasion and immune escape. Citation Format: Riya Chhabra, Alfred Kao, Suravi Bajaj, Reena Ding, Daniel John, Wei Tse Li, Jessica Y. Wang-Rodriguez, Weg M. Ongkeko. Spatial neuroimmune crosstalk driving perineural invasion in head and neck squamous cell carcinoma [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 5559.

Abstract Introduction: Advanced cutaneous melanoma shows substantial heterogeneity in clinical outcomes, even among patients classified within the same clinical stage. Understanding the molecular drivers underlying this heterogeneity is critical for advancing treatment strategies. Previous studies have identified distinct melanoma transcriptomic states - Tirosh et al. and Balderson et al. have agreed on a four-subtype model defining “Undifferentiated”, “Neural Crest”, “Transitory”, and “Melanocytic” states. However, their biological and clinical relevance remains unclear. Here we profiled in-transit melanoma (ITM) using digital spatial RNA profiling to associate melanoma transcriptomic states with overall survival (OS) and acral melanoma (AM) status. Methods: Digital spatial profiling (Nanostring GeoMx Whole Transcriptome Atlas) was performed across a tissue microarray constructed from patients with ITM diagnosed from 1990-2020. After filtering poor quality areas of interest (AOIs), we processed the data using noise correction and quantile normalization. We applied Principal Component Analysis (PCA) to define melanoma subtype signatures by ranking genes contributing to each of the first four PCs. We performed gene set enrichment analysis using GSEA with significance defined as an adjusted p-value < 0.05. Association between gene or gene-set expression and OS was evaluated using optimal expression cutoffs to define high and low groups, with a minimum group size of 20%. Cox proportional hazards models were then used to assess survival differences between groups. Results: We analyzed a cohort of 84 patients (116 AOIs) with ITM passing QC. PCA of transcriptional profiles revealed distinct transcriptional states. The PC1 axis differentiated Transitory from Undifferentiated melanoma, PC2 reflected immune cell infiltration, PC3 corresponded to stromal cells and Neural crest-like melanoma, and PC4 associated with Melanocytic melanoma. We derived gene sets from the PCA results and associated the expression of each gene set with OS. Across a cohort of treatment-naïve ITM, high expression of the melanocytic state conferred a median overall survival difference of 7.59 years (melanocytic ‘high’=5.16 years vs ‘low’=12.75 years, log-rank p=0.0024) and independently associated with poor survival in multivariate analysis. AMs showed higher melanocytic state gene expression compared to non-acral cases. These findings were validated in external datasets, supporting that the melanocytic state predicts poor prognosis. Conclusion: The melanocytic transcriptional state was independently associated with worse overall survival in patients with metastatic melanoma and was enriched in acral melanoma, suggesting that assessment of the melanocytic state may have value for clinical risk stratification. Citation Format: Ziyin Huang, Kristen E. Rhodin, Rami Al-Rohil, Viviana Geron, Arul M. Chinnaiyan, Margaret H. O'Connor, Christina Vadala Angeles, Smita K. Nair, Georgia M. Beasley, Matthew K. Iyer. Melanocytic transcriptional state is an independent marker of survival in metastatic melanoma [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 4146.

Abstract The extensive heterogeneity in the tumor immune microenvironment is a problem plaguing immune-based treatment failures for solid tumors because it prevents defining the specific vulnerabilities of the various cells to develop treatments with greater precision. Myeloid-derived suppressor cells (MDSCs) are a heterogenous collection of immunosuppressive monocytes and granulocytes (neutrophils) whose link to poor cancer survival is not well understood. Most MDSCs exhibit features of immature granulocytes (Gr-MDSCs) and exert their T cell suppression by secreting reactive oxygen and nitrogen species (RONS). Gr-MDSCs can survive for days by recruiting mechanisms to protect protein translation despite generation of RONS. One MDSC subtype of interest is defined by their polarization in response to type 1 interferon (IFNα) that we previously identified in chronic Helicobacter-infected mice with gastric metaplasia, suggesting that a Helicobacter-infected stomach polarizes neutrophils to acquire an immunosuppressive phenotype. We hypothesize that IFNα initiates neutrophil reprogramming by increasing protein synthesis required to alter their metabolic activities as they acquire the Gr-MDSC phenotype. We performed scRNA-Seq on biopsies from 5 groups of patients referred for endoscopy and showed no gastric abnormalities, were H. pylori+ (Hp), had intestinal metaplasia (IM) or gastric adenocarcinoma (GAC) and identified that the IFN-polarized MDSC population initially present in IM increased substantially in GACs but was not detected in normal and Hp-infected patients, confirming the polarization of IFN-polarized Gr-MDSCs once IM appears. We showed that the IFN-polarized Gr-MDSCs re-programmed from neutrophils in vitro and required induction of protein translation. To analyze translation, we treated primary cultures of granulocytes from mouse bone marrow or human PBMCs with IFNα and quantified protein translation by flow cytometry after incubating the cultures with 20µM O-propargyl-puromycin (OPP) and proliferation with EdU. In vivo analysis was performed by administering OPP to mice before euthanization. In the in vitro neutrophil cultures, about 28% of the polarized neutrophils were CD11b+Gr-1+ MDSCs, and 85% of these Gr-MDSCs induced their protein synthesis coincident with increased proliferation. scRNA-Seq analysis of both mouse and human gastric tissues demonstrated induction of mediators of the unfolded protein response (UPR), e.g., CHOP. In summary, polarization of Gr-MDSCs by type 1 IFNs from neutrophils requires induction of protein translation, revealing possible metabolic pathways that may be targeted to reprogram the immunosuppressive tumor microenvironment. Citation Format: Juanita L. Merchant, Lin Ding. IFNα polarizes granulocytic MDSCs from neutrophils by inducing protein translation [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 6082.

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) requires the identification of new therapeutic targets to improve treatment outcomes. This study investigated the biological significance of Microtubule Affinity-Regulating Kinases (MARKs), a family of serine-threonine kinases that regulate microtubule-associated proteins (MAPs). Because MARK activity influences cell structure, intracellular transport, and proliferation, these kinases may represent promising therapeutic targets. Materials and Methods: Protein expression levels of MARK family genes were evaluated in 20 human HNSCC cell lines. Cisplatin and docetaxel-resistant models were established by gradually escalating cisplatin and docetaxel concentrations up to 4 μM and 15 nM, respectively in CA9-22 and YD-8 cells. Cell viability was determined using WST assays. CRISPR/Cas9 genome editing was employed to generate MARK2/3/4 knockout cell lines. In addition, the anticancer efficacy of several newly developed MARK2/3/4 hits was assessed. Results: MARK2/3/4-knock-out cells demonstrated significantly reduced proliferation and impaired matrigel invasion compared with wild-type controls. MARK kinase loss was associated with suppression of the MAPK- and PI3K-AKT signaling pathways. Because MARK proteins are known to interfere with MST/SAV and LATS complex formation through phosphorylation-dependent mechanisms, we further showed that MARK4 depletion reduced nuclear YAP/TAZ localization and decreased expression of YAP/TEAD transcriptional targets. Collaborative screening identified multiple candidate molecules for MARK-specific inhibition at the hit-compound stage; notably, one lead compound exhibited strong antiproliferative activity in HNSCC cells, including cisplatin-resistant models. Conclusions: MARK proteins function as negative regulators of the HIPPO kinase cascade, thereby enhancing YAP/TAZ oncogenic activity in HNSCC. Loss of MARK function reduces tumorigenic phenotypes, supporting MARKs as promising therapeutic targets for future drug development. Citation Format: Se Eung Oh, Hwan Jung Lim, Seong Jun Park, Sang Uk Han, Jee Hung Kim, Seo Young Lee, Hei-Cheul Jeung. MARK2/MARK3/MARK4 kinases are therapeutic targets for human head and neck squamous cell carcinoma with co-dependencies of YAP-TEAD pathway [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 327.

To analyze gender representation across a hierarchy of speaking roles at major international ophthalmology conferences and quantify disparities in high-prestige invited roles vs merit-based submitted roles. Annual meetings of seven international ophthalmology societies (AAO, ASCRS, ESCRS, UKISCRS, AUSCRS, BRASCRS, ICO) from 2019 to 2024. Retrospective, cross-sectional study. A total of 40,940 speaker slots were analyzed. Gender was determined using a validated protocol combining API analysis and manual verification. Roles were classified into a three-tiered hierarchy: Tier 1 (High Prestige: named lecturers, keynotes, awards), Tier 2 (Invited Faculty), and Tier 3 (General Presenter: submitted papers). Multivariable logistic regression assessed the association between gender and role prestige. Women held 5,397 (36.6%) of Tier 3 (submitted) roles but only 45 (25.6%) of Tier 1 (high-prestige) roles. In multivariable analysis adjusted for conference and year, women had significantly lower odds of occupying a high-prestige role compared with a submitted role (adjusted odds ratio, 0.59; 95% CI, 0.42-0.84; P = .003). A "glass podium" effect was observed, with women's representation decreasing as role prestige increased. Despite robust participation in submitted research, women remain underrepresented in the most visible, career-advancing roles. This disparity supports consideration of more objective and structured approaches to speaker invitation and role allocation in invited conference programming.

Abstract The acidic microenvironment (AME) is a defining metabolic hallmark of solid tumors and a key driver of coevolution between cancer cells and the surrounding stroma. AME arises primarily from hypoxia in avascular tumor regions and from the Warburg effect in normoxic habitats, resulting in sustained extracellular acidosis that imposes strong selection pressure on all cellular populations within the tumor ecosystem. Cells adapt to this stress either through Darwinian selection of pre-existing variants or through non-Darwinian phenotypic plasticity, leading to metabolic and transcriptional heterogeneity that supports tumor progression. The AME profoundly influences multiple stromal components - including collagen, fibronectin, fibroblasts, macrophages, and immune cells - thereby remodeling the tumor habitat and promoting invasion and metastasis. To investigate the mechanisms of stromal adaptation, we performed single-cell MALDI imaging to profile metabolic and lipidomic heterogeneity in fibroblasts exposed to acidic conditions for short and long durations, representing stages of acclimation and adaptation. Acid-exposed fibroblasts displayed altered sphingolipid and ceramide metabolism, suggesting metabolic plasticity as a key mechanism of survival under low pH.We then co-cultured acid-adapted fibroblasts with cancer cells in 3D spheroids and analyzed them using spatial metabolomics and lipidomics. Acid-adapted fibroblasts induced distinct metabolic niches that enhanced cancer cell invasiveness and ecosystem-level cooperation. To extend our findings to patient samples, we used MACSima cyclic immunofluorescence and MALDI imaging on DCIS tissues to spatially correlate metabolic changes with stromal reprogramming. Acid-exposed regions exhibited fibroblast activation markers (α-SMA, FAP, vimentin) and histone variant enrichment, linking metabolic stress to epigenetic remodeling. Finally, we developed a mathematical model integrated with experimental data as a tool to test hypotheses of DCIS progression based on stromal adaptation and metabolic remodeling. The model incorporated tumor tissue histology images to simulate spatial metabolic interactions and fibroblast-cancer coevolution under acid stress, and predicted that fibroblast plasticity accelerates tumor expansion. Together, our findings reveal that acidosis-induced metabolic and epigenetic reprogramming of fibroblasts drives stromal activation, niche remodeling, and DCIS progression, providing an evolutionary framework to target tumor acidity and its adaptive consequences. Citation Format: Emma R. Downey, Ana M. Forero Pinto, Katarzyna A. Rejniak, Mehdi Damaghi. Acidic microenvironment as a driver of cancer-stroma coevolution: Integrating experimental and mathematical models of fibroblast adaptation and metabolic remodeling [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 781.

Abstract Many cancers have inherent defects in DNA damage response (DDR) which influence their sensitivity to tumor-targeting therapies. Examples include increased activity of immunotherapies in cancers with mismatch-repair deficiency and sensitivity to PARP inhibitors and platinum-based therapies in the context of homologous recombination (HR) deficiency. However, we are currently unable to reliably determine which DDR defects are present in a given cancer sample, which severely limits our ability to exploit these therapeutic vulnerabilities. Structural variants (SVs), or genomic rearrangements formed as a product of aberrant double strand break repair, hold promise as biomarkers of DDR state. Indeed, SVs affect a larger proportion of the cancer genome than any other form of genetic alteration and have features that reflect their mechanism of formation. Here, we develop QuantHDP, a novel computational method for detecting SV signatures which leverages complex modeling of genetic features to distinguish between cancers with different DDR alterations and potentially identify clinically relevant biomarkers. QuantHDP models SV features with probability distributions that reflect our knowledge of the biological mechanisms that generate them, accounts for expected differences in signature content by cancer type, and automatically infers the number of signatures present in a given dataset. In addition to recapitulating multiple previously established associations with defects in DDR - including signatures of BRCA1, BRCA2, and CDK12 alterations — we also uncover novel signatures that warrant further investigation. Citation Format: Gregory Raskind, Youyun Zheng, Anthony Zhao, Julia Sun, Simona Dalin, Siyun Lee, Chunyang Bao, Antonia Kowalewski, Ron Solan, Sam Wiseman, Samantha Van Seters, Saveliy Belkin, David I. Heiman, Chip Stewart, David Lehotzky, Vasuki Narasimha Swamy, Brian P. Danysh, Luis Antonio Corchete Sanchez, Andrew D. Cherniack, Haruna Tomono, Gengchao Wang, Xavi Loinaz, Zachary Everton, Gang-Hee Lee, Won-Chul Lee, Hansol Park, Ryul Kim, Young Seok Ju, Gad Getz, Esther Rheinbay, Rameen Beroukhim. Structural variant signature discovery across >8,000 TCGA whole genomes using QuantHDP [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 7274.

Abstract Background: Prostate cancer (PCa) remains a highly lethal disease due to the rapid emergence of neuroendocrine (NE)-like variants from adenocarcinoma. It is predicted that de novo NEPC will emerge from 17% of localized PCa, while treatment-related NEPC will account for 20% of advanced PCa. Clinically, NEPC patients frequently harbor visceral metastasis (62%, P<0.001) compared with CRPC patients (24%), with poor overall survival of 12-17 months. Thus, exploring the unknown mechanism underlying cell-state transition from adeno to NEPC will help identify new targets to overcome lineage plasticity and improve PCa patient survival. Methods: We developed a novel indolent and metastatic PCa mouse model by overexpressing cMyc and knocking out Pten with/without Mutant p53 (R172H). Global transcriptional profiling was performed in indolent and aggressive mice and ASPORIN (ASPN) knockdown (KD) to identify differential gene expression, biological, and pathway analyses. ASPN ectopic overexpression (OE) and KD clones confirmed ASPN biological function and on-target proteins/genes using RT-PCR and western blot analyses. ASPN in vitro function was analyzed in the Incucyte® live imaging system, colony growth assay, and proliferation assays. Results: The novel Ptenfl/fl; Hi-Myc; Trp53R172H/+; Rosa-26; PB-Cre4+ (PCTPLuc) mouse exhibits phenotypic resemblance to PCa visceral metastasis to the lung, liver, inguinal lymph node, and intestine with poor survival as compared with age-matched indolent Ptenfl/fl; Hi-Myc; Rosa-26Luc; PB-Cre4+ (PCPLuc). Unbiased RNA-Seq analysis of PCa adenocarcinoma tissues from PCTP mice revealed a significant association between unique extracellular matrix (ECM) protein clusters and liver and lung metastasis. Asporin (ASPN) emerged as the top differentially expressed ECM protein among the top 25 genes. Consistently, ASPN knockdown and overexpression were directly associated with PCa cellular phenotypes of proliferation and colony growth. Mechanistically, ASPN modulation impacts pERK, CyclinD3, EMT proteins, and a neuroendocrine-like phenotype, as well as cell differentiation-related transcriptomes such as cellular retinoic acid-binding protein 2 (CRABP2), along with Chromogranin A (CHGA), NeuroD1, and INSM1. Using a publicly available TCGA database, we observed a significant overexpression of ASPN and CRABP2 in PCa tissues (N=497) relative to normal (N=52) (P<0.001). Finally, ASPN ectopic overexpression in immortalized RWPE-1 cells confirmed an aggressive in vitro phenotypes. Conclusion: For the first time, we explored the influence of ASPN/CRABP2 and other signaling involved in luminal epithelial to neuroendocrine differentiation and validated CRABP2 as a novel target to prevent NEPC transdifferentiation. Our findings support ASPN’s new role, mechanism(s) and as a target for PCa and other visceral metastasis disease. Citation Format: Parthasarathy Seshacharyulu, Shobhit Lall, Sushanta Halder, Sakthivel Muniyan, Zahraa Wajih Alsafwani, Ramakanth Chirravuri-Venkata, Moorthy P. Ponnusamy, Surinder K. Batra. Novel ASPORIN/CRABP2 axis drives cell-state plasticity from adenocarcinoma to neuroendocrine-like prostate 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 5573.

Abstract Integrin beta-6 (ITGb6) is a heterodimeric, cell-surface glycoprotein highly expressed in multiple solid tumor indications, including non-small cell lung cancer, esophageal cancer, head and neck cancers, breast and gastric cancers. Its expression has been associated with pro-tumorigenic activities including proliferation, migration, and invasion. ITGb6 has restricted expression in adult tissues, making it a highly promising target for cancer therapy using antibody drug conjugates (ADCs). STRO-006 is an investigational ADC composed of an ITGb6 targeted human IgG1 antibody conjugated to exatecan, a topoisomerase 1 inhibitor, via a b-glucuronidase-cleavable linker. The anti-ITGb6 antibody was discovered from a Fab ribosome display library using Sutro’s XpressCF+® system. The drug-linker is functionalized with dibenzylcyclooctyne (DBCO) and allows rapid and selective site-specific conjugation to the azide-containing non-natural amino acid p-azidomethylphenylalanine (pAMF) incorporated into the antibody (Ab) sequence using XpressCF+®. This site-specific conjugation is highly efficient, resulting in a well-defined, homogeneous ADC with a drug-antibody ratio (DAR) of 8. The STRO-006 antibody binds specifically to the alpha-v beta-6 heterodimer and has high affinity to human and cynomolgus ITGb6. It demonstrates rapid and efficient internalization, ideal for an ADC mechanism of action. Importantly, the STRO-006 antibody does not compete for LAP binding and therefore does not interfere with TGFb signaling. STRO-006 is optimized for a stable pharmacokinetic profile and exhibits extended half-life of ∼7 days, low clearance of ∼5 mL/d/kg and maintains a stable DAR over the course of 21-days in non-human primates. In preclinical studies, STRO-006 demonstrates potent anti-tumor activity at clinically relevant dose in both PDX and xenograft models of non-small cell lung, head and neck squamous cell and pancreatic carcinomas. STRO-006 demonstrates a favorable safety and pharmacokinetic profile in non-human primates. These results suggest that STRO-006 is a promising candidate for the treatment of multiple carcinomas and supports further clinical investigation. A Phase 1, first-in-human study is planned to assess the safety and activity of STRO-006. Citation Format: Kshama A. Doshi, Stephanie Armstrong, Eunice Kim, Dan Shen, Sihong Zhou, Rhoneil Pena, Robert Yuan, Mark Armanini, Brian Vuillemenot, Xiaofan Li, Guifen Xu, Krishna Bajjuri, Miao Wen, Jeff Hanson, Cuong Tran, Amandeep Gakhal, GARRETT GROSS, Gang Yin, Werner Rubas, Genevive Hernandez, Daniel Calarese, Hans-Peter Gerber, Alice Yam. STRO-006: An Integrin beta-6-targeting ADC demonstrates favorable safety profile and potent antitumor activity in preclinical solid 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 1713.

Abstract Background: The tumor microenvironment (TME) plays a pivotal role in cancer progression, immune evasion, and therapeutic response. Understanding the spatial organization and functional states of cells within the TME is essential for advancing immuno-oncology and precision medicine [PMID: 40102282]. However, simultaneous visualization of secreted molecules and cellular phenotypes in situ remains a major challenge in the spatial biology field [PMID: 39930476]. Here, we employed an automated hyperplex multiomics assay to simultaneously detect RNA and protein expressions to spatially map cell phenotypes and their functional states in the TME of multiple cancer types. Methods: We examined a formalin-fixed paraffin-embedded tissue microarray (TMA) comprising various human cancer types: prostate, lung, breast, colorectal, melanoma, and lymphoma. A TMA section was stained and imaged on the COMET™ platform, integrating RNAscope™ HiPlex Pro for transcript detection and sequential immunofluorescence (seqIF™) for proteomic analysis [PMID: 22166544; 37813886; 41065276]. Image was analyzed using HORIZON™ software to extract single-cell and spatial features. Results: The automated multiomics approach enabled the concomitant in situ detection of over 100 biomolecular targets, including 12 transcripts and more than 90 proteins on the same section. High-resolution spatial profiling of the TME allowed accurate mapping of cancer, stromal, vascular and diverse immune cell subsets. It further revealed key molecular features associated with tumor-suppressor or proto-oncogene activity, including markers of proliferation, apoptosis, and immune checkpoint regulation. Concurrent detection of cytokine and chemokine transcripts highlighted localized immune signaling and cell-cell communication within tumor and stromal compartments. Conclusions: This multiomics workflow offers a powerful tool for in-depth characterization of the TME across multiple cancer types, while significantly reducing sample consumption. Spatial profiling provides new opportunities to dissect the tissue architecture and immune dynamics to identify functional cell states and interactions to be exploited in immunotherapy and personalized medicine. Citation Format: Marion Bonnet, Cansaran Saygili-Demir, Kim Handel, Pedro Machado, Alix Failletaz, Debia Wakhloo, Anushka Dikshit, Maria Giuseppina Procopio, Saska Brajkovic. Automated multiomics assay with over 100 biomarkers for in-depth spatial profiling of the tumor microenvironment in multiple cancer types [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 4969.

Abstract Background: PD-1/PD-L1 antibody-based therapies have demonstrated tremendous success in the treatment of a variety of cancers. However, these antibody drugs are associated with several disadvantages, such as weak tumor penetration, immune-related adverse events and emergence of anti-drug antibodies. Here, we report the discovery and characterization of ALG-094295 as a highly potent and orally bioavailable small molecule PD-1/PD-L1 inhibitor that binds to PD-L1 and promotes PD-L1 dimerization, internalization and degradation, offering a different mechanism of action with potential advantages over PD-1/PD-L1 antibody therapeutics. Methods: The interaction of PD-1/PD-L1 and PD-L1 dimerization were assessed by AlphaLISA®. Cellular activity was measured using PD-1 expressing Jurkat NFAT luciferase T cells and CHO-hPD-L1 cells. In vivo PD-L1 target engagement, tumor growth inhibition and tumor infiltration of T-cells were assessed in a humanized-PD-L1 MC38 subcutaneous tumor mouse model. In vitro ADME tox profile was established using standard assays. Pharmacokinetic (PK) studies were performed with rat, dog and cynomolgus monkey. Results: ALG-094295 demonstrated inhibition of PD-1/PD-L1 interaction at sub-nanomolar concentrations and induced PD-L1 dimerization. In vitro studies showed that ALG-094295 activated T cells with approximately ten times greater potency compared to INCB086550, an orally administered small molecule PD-L1 inhibitor that has demonstrated clinical responses in a phase I trial. Furthermore, treatment of CHO-hPD-L1 cells with ALG-094295 resulted in internalization and reduction of PD-L1 protein levels. In ex vivo human PBMC assays, ALG-094295 demonstrated PD-L1 target engagement, T cell activation and immune cell mediated tumor cell killing. In a humanized PD-L1 MC38 mouse model, a single oral dose of ALG-094295 (5 mg/kg) achieved PD-L1 target engagement comparable to INCB086550 (150 mg/kg PO). Daily oral dosing of ALG-094295 (50 or 150 mg/kg) in humanized PD-L1 MC38 mice over 21 days resulted in tumor growth inhibition equivalent to twice-weekly administration of durvalumab (10 mg/kg IV), with tumor size correlating with increased CD8+ T-cell infiltration. ALG-094295 demonstrated no in vitro liabilities for CYP450 inhibition or induction mediated drug-drug interactions, off target toxicity, cardiovascular safety, or genotoxicity. Preclinical in vivo PK data suggests once-daily oral dosing is feasible in humans. Conclusion: ALG-094295 is a highly potent and orally bioavailable small molecule PD-1/PD-L1 inhibitor that promotes PD-L1 dimerization, internalization and degradation. ALG-094295 has the potential to overcome some limitations of antibody-based therapies due to potent PD-L1 blockade, oral delivery and novel mechanism of action. Citation Format: Heleen Roose, Kristina Rekstyte-Matiene, Sarah Stevens, Kusum Gupta, Sandra Chang, Nour Fayyad, Cheng Liu, Vladimir Serebryany, Lillian Adame, Kha Le, Antitsa Stoycheva, Dinah Misner, Lawrence M. Blatt, Sushmita Chanda, David B. Smith, Julian A. Symons, Andreas Jekle, Tongfei Wu. Preclinical Characterization of ALG-094295, a highly potent and orally bioavailable small molecule PD-1/PD-L1 inhibitor targeting dimerization, internalization and degradation of PD-L1 [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 2818.

Abstract Venetoclax based combination therapy is utilized as the first-line treatment for elderly acute myeloid leukemia (AML) patients with short remission time due to resistance and relapse. Previously, we reported that artesunate enhanced venetoclax-induced apoptosis by promoting NOXA-mediated degradation of Mcl-1. By employing a chemical conjugation approach, we linked dihydroartemisinin (DHA) to venetoclax using a two-carbon methylene spacer, yielding the conjugate A1. A1 maintains Bcl-2 inhibitory activity and overcomes Mcl-1/Bcl-xL-mediated resistance. However, due to its large molecular size, A1 has limited bioavailability and solubility. To address these limitations, we incorporated various polyethylene glycol (PEG) units between venetoclax and DHA, generating conjugates A18-A20. These derivatives exhibited approximately two-fold greater solubility than A1, and more potent activity to inhibit colony formation of U937 cells in soft agar assays. Moreover, A19 and A20 significantly suppressed tumor growth in vivo. The tumor growth inhibition rates for A19 and A20 (75.6% and 65.8%, respectively) were significantly higher than that of venetoclax alone (33.9%). We further modified the PEG backbone of A20 by incorporating nitrogen-containing polar groups, resulting in compounds A21-A23. These modifications led to further increased aqueous solubility and colony-forming inhibitory activity. These novel conjugates represent promising next-generation venetoclax derivatives capable of overcoming resistance. Citation Format: Jingyi Zhang, Linghui Hou, Zhenwei Zhang, Samuel Waxman, Linxiang Zhao, Yongkui Jing. Optimizing the linker of venetoclax-artemisinin conjugates to improve water solubility and antileukemia effects [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 7087.

Abstract Accurate measurement of regulated cell death (RCD)—including apoptosis, necroptosis, and necrosis—is critical for oncology drug development and mechanism-of-action studies. Conventional fluorescence assays introduce phototoxicity, labeling bias, and incompatibility with long-term or high-frequency pharmacodynamic monitoring. We developed a fully label-free platform that integrates 3D holotomography (HT) and deep learning to classify RCD phenotypes directly from intrinsic refractive-index (RI) signatures, enabling non-perturbative, mechanism-aware drug-response biomarkers.HeLa cells were induced into apoptosis, necroptosis, or necrosis using canonical biochemical triggers, with fluorescence markers (Annexin V, PI, Hoechst) used solely for ground truth. 3D RI tomograms acquired by HT-X1 Plus were converted to 2D maximum-intensity-projection (MIP) patches to train an ImageNet-pretrained CNN to classify five states (live-control, live-treated, apoptosis, necroptosis, necrosis) using a sliding-window/majority-vote strategy. Temporal concordance was evaluated through synchronized HT–fluorescence time-lapse imaging and flow cytometry. For subtle drug-induced phenotypes (doxorubicin, cisplatin), performance of full 3D volumetric models was compared with 2-D projections to assess the necessity of depth information. Cross-cell-line robustness was tested on A549 cells with minimal fine-tuning.The five-state classifier achieved 99.3% accuracy on held-out HeLa datasets, with misclassifications limited to the apoptosis–necroptosis boundary. HT-based predictions identified early necroptotic transitions 2–4 hours before Annexin V/PI fluorescence, and population-level dynamics closely matched flow cytometry, establishing an earlier, dye-free pharmacodynamic window. In drug-response experiments, 3D volumetric models outperformed all 2D approaches, capturing spatially heterogeneous, mechanism-rich morphological signatures (76–88% accuracy in 3D vs. 50–55% in 2D MIP and 0% in SUM projections). The HeLa-trained model generalized poorly to A549 cells initially (50.4% accuracy), but small-data fine-tuning restored near-perfect performance, demonstrating practical assay portability across cancer cell types.Holotomography-based AI provides a fully label-free, segmentation-free, real-time biomarker for distinguishing RCD pathways and quantifying early drug responses with high accuracy. The platform detects necroptosis hours earlier than biochemical assays, resolves subtle drug-induced morphologies, and adapts rapidly to new cell types. These capabilities position HT-AI as a scalable pharmacodynamic tool for mechanism-of-action profiling, cytotoxicity testing, and high-content oncology drug discovery, enabling longitudinal, non-destructive phenotyping beyond fluorescence-based methods. Citation Format: Minwook Kim, Park Weisun, Geon Kim, Sanggeun Oh, Juyeon Park, Jihwan Yu, Hyun-Suk Min, Sumin Lee, Won Do Heo, YongKeun Park. Label free identification of cancer cell death pathways via holotomography and deep learning as an early pharmacodynamic biomarker [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 4669.

Abstract Cold tumors evade immunotherapy through limiting T cell priming inside draining LN or infiltration into TME. Here, we have developed sequential strategies to overcome the limitation. Firstly, we have developed new mRNA vaccine expressing membrane cytokines with shared or mutated antigens to more and better prime tumor specific T cells (TST) without toxicity. Secondly, we generated tetramer forms of NGR that fusing into Fc-pro-IL2 that selectively targeted CD13 enriched on TME for more IL-2 to rejuvenate dysfunctional T cells. Unexpectedly, cis-delivery of IL-2 on CD13 on tumor vessels can bridge the cross-talk between tumor vessels and TST. Activated T cells can remodel tumor vessels to allow more infiltration. To sustain their anti-tumor activities, we have sequentially delivered tumor-activating cytokines (pro-IL2) guided by anti-PD-1 antibody (anti-PD-1-pro-IL2) or radiation-activating TLR agonists to help DC-T cell interaction inside TME to sustain T-cell effector function. Together, we have sequential strategies converting cold to hot tumors and then simultaneously reinvigorates TIL to overcome the limitations of current immunotherapies. Citation Format: Yang-Xin Fu, . The Immune strategies to convert cold to hot tumors and overcome resistances [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 4245.

Abstract Background: Overexpression of ABCC1 is associated with poor overall survival across many cancers and has been associated with decrease response to chemotherapy. Our in vivo and in vitro studies have shown that ABCC1 expression is higher in laryngeal squamous cell carcinoma (LSCC) from individuals with African heritage. Here, we further investigate ABCC1 as a potential prognostic and predictive marker in laryngeal cancer health outcomes. Methods: ABCC1 gene expression was assessed in tumor tissue from 32 advanced-stage LSCC patients from differing populations via RT-PCR. Immunohistochemistry (IHC) for ABCC1 protein expression was performed on tissue microarrays (TMAs) generated from 61 paraffin-embedded tissues of advanced stage LSCC from patients of differing heritages. Machine learning based digital pathology software (QuPath) was used to semi-quantitate ABCC1 IHC protein expression as H scores. Age, treatment, self-reported heritage, N stage, tumor histologic grade, and ABCC1 H-scores were evaluated as predictors of patient mortality risk via multivariable Cox proportional hazards (CPH) model. The median Cox risk score (cutoff > 0.971) was used to stratify patients into low and high mortality risk groups. The relationship among the clinical features and ABCC1 in low and high mortality risk groups was explored by Chi Square analysis. Overall survival was assessed using the Kaplan-Meier method. Results: LSCCs from patients with African heritage demonstrated a 2.2-fold elevation in ABCC1 gene expression and higher ABCC1 protein expression (H-score) compared with the reference population (p < 0.05). Controlling for treatment, African heritage patients with low ABCC1 H-scores had a high mortality risk (p < 0.05), and patients from the reference population with metastatic disease and high ABCC1 H-scores had a low mortality risk (p < 0.05). Overall, high-risk patients had significantly worse overall survival (HR = 2.46, 95% CI [1.27-4.74], log rank p < 0.01) and high mortality risk was associated with low ABCC1 H-scores and regional metastatic disease (p < 0.05). Conclusion: Although LSCC patients with African heritage have higher ABCC1 expression compared with the reference population, low ABCC1 H-scores carry a higher mortality risk in this group. LSCC patients from the reference population with high ABCC1 H-scores have reduced mortality risk despite having regional metastatic disease. Future studies will involve exploring mechanistic implications of ABCC1 expression on treatment outcomes across differing heritages. Citation Format: Christina Gobin, Matthew Chang, Jai Walker, Kristianna Fredenburg. Evaluating ABCC1 as a potential prognostic biomarker in laryngeal cancer health outcomes [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 2386.

Abstract Background: Pancreatic cancer (PDAC) is nonresponsive to conventional therapies and poor survival is the norm. A small percentage of malignant cells undergo epithelial-mesenchymal transition to evolve into pancreatic cancer cells stem cells (PCSCs) that evade therapy and subsequently give rise to residual/recurrent tumors and also drive therapy resistance. While radiotherapy (RT), is a cornerstone of PDAC management, it promotes EMT via activation of TGF-β, NF-kB and Akt/GSK-3β pathways. TIM-3 (HAVCR-2) a cell surface marker traditionally designates exhausted T-cells. However TIM-3 overexpression is being increasingly reported in solid tumors. We report RT induced TIM-3 overexpression in PDAC and propose that RT triggered TIM-3, on pancreatic cancer cells could serve as a novel target in selectively eliminating PCSCs. Methods: GEO datasets were analyzed with the Seurat package in R to evaluate TIM-3 expression across tissue types (benign pancreatic tissue, non metastatic primary PDAC/Pm0, primary of metastatic PDAC/Pm1 and matched liver metastases/Lm). TIM-3 expression between treatment naive and chemo-RT treated tumors was also compared. In vitro, murine (Panc02-ova, KPC) and human (PANC-1, MIA PaCa-2) pancreatic cancer cells were treated with increasing doses of RT alone or in combination with NRF-2 / ATM inhibitor to either simulate a high oxidative stress environment or inhibit DNA repair pathways. Following treatments, TIM-3 expression in these samples were evaluated using RT-qPCR, western blotting, flow cytometry and immunofluorescence. In vivo, Panc-02 xenografts in C57BL/6 mice were irradiated (10Gy RT at 320Kv & 13.2 mA). Tumors were collected, 24 hrs post RT, and dissociated. Single cells thus obtained were assessed for the expression of TIM-3, using flow cytometry. Results: Advanced stages of cancer (Pm1 and Lm tumors) demonstrated greater TIM-3 expression compared to Pm0 tumors. Further, increased TIM-3 expression was noted in chemo-RT treated tumors compared to untreated tumors. In vitro, RT dose dependent increase in TIM-3 mRNA and protein levels was recorded and cells subjected to 10Gy RT showcase maximal TIM-3 expression (Control < 3Gy < 6Gy < 10Gy). Addition of NRF2 inhibitor or ATM inhibitor before exposing cells to RT further increased TIM-3 levels.The TIM-3+ cells exhibited higher CD24+ and CD44+ positivity, indicating its association with PCSC phenotype. Conclusion: Our results indicate that TIM-3 can be employed as a specific marker to designate PCSCs in addition to the classical stem cell phenotype (ESA+ CD24+ CD44+). Increased mRNA, total protein and TIM-3 levels post RT are possibly driven by elevated oxidative stress in addition to RT induced ds-DNA breaks. The novel role of TIM-3 as a PCSC marker, establishes it as an ideal candidate for targeting both immune-suppressive T cells and therapy resistant PCSCs. Citation Format: Prapannajeet Biswal, Prudhvi Chand Mallepaddi, Bhoomika Murvekere Lakshmisha, Tuyet Ngoc Tra, Sai Kumar Samala, Khadijeh Koushki, P.M. Quan Mai, Arjun Vasan, Gabrielle Krouse, Yuri Mackeyev, Lydia WT Cheung, Sunil Krishnan, Geraldine Vidhya Vijay. TIM-3 as a prospective target to eliminate pancreatic cancer stem cells [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 4820.

Abstract Introduction: AML is a molecularly heterogeneous disease that is classified by recurrent cytogenetic abnormalities and gene mutations. Recent studies have shown divergent frequencies of several genetic aberrations depending on genetic ancestry and self-reported race/ethnicity, highlighting the need to broaden sequencing efforts to include more diverse pts. Methods: We performed paired tumor/normal whole exome sequencing (WES) and transcriptome sequencing on 271 ancestry and/or ethnically diverse pts [including 100 African American (AA) and 71 self-identified Hispanic pts; CALGB/Alliance], and a validation cohort of 45 AA pts (University of Pennsylvania). Results: We identified >20 genes to be mutated in 3-8% of pts that were not seen in previous sequencing efforts of predominantly non-Hispanic White/European ancestry (EA) pts. Notably, variants in genes encoding Rho-GTPase regulatory proteins (ARHG family, belonging to the RAS gene superfamily) were identified in 12% of pts, placing these genes in the top 5 of recurrently mutated genes in this pt cohort. This frequency was confirmed in the second cohort of AA pts (n=7/45, 15%). In contrast, analysis of 805 EA adults with WES data (BeatAML 2022) and 877 pediatric AML pts (TARGET) found ARHG gene family variants in 26/805 (3%) and 17/877 (1.9%) of EA AML pts, respectively. With a median age of 41y, ARHG-mutated(m) pts tended to be younger (P=.16) and more often diagnosed with core-binding factor AML (39% vs 19%, P=.02). ARHG mutations frequently co-occurred with NRAS and FLT3 mut (each found in 35% of ARHGm pts). Notably, survival of ARHGm pts was poor, with a median overall survival (OS) of <12 months, thereby mirroring OS of the 2022 European LeukemiaNet (ELN) Adverse risk group. Within the 2022 ELN Favorable risk group in the ancestry diverse cohort, ARHGm pts had shorter OS than ARHGwt pts (P=.02). The clinical outcome was especially poor in young adolescents and adults (AYA, 18-39y) (mut vs wt; 3y disease-free survival, 10% vs 50%, P<.001; 3y OS, 20% vs 55%, P=.008). RNAseq of 17 ARHGm pts showed transcriptomic RAS pathway activation, with 52% displaying a RAS-associated signature, also in the absence of other RAS mutations. Furthermore, bulk transcriptomic analyses of 1250 AML pts identified 120 predicted RAS signature genes, with the upregulated genes being enriched in metallopeptidases, MAP kinase phosphatases, and ARHG genes. Conclusion: We identified mutations in ARHG family genes as frequent yet thus far unrecognized RAS pathway activators in AML associated with poor survival that are not yet included in clinical testing panels. Their lack of recognition is likely due to the heterogeneity of mutationally affected ARHG family genes, enrichment in AYA pts and the high frequency in pts of non-European ancestry, both of which are pt populations that were underrepresented in previous sequencing efforts. Citation Format: Ethan Hamp, Lorenz Oelschläger, Bailee N. Kain, Deedra Nicolet, Krzysztof Mrozek, Katherine E. Miller, Audrey Bollas, Michael C. Walker, Christopher J. Walker, Jill Buss, Andrea Laganson, Andrew J. Carroll, William G. Blum, Bayard L. Powell, Geoffrey L. Uy, Wendy Stock, Marina Y. Konopleva, Richard M. Stone, John C. Byrd, Martin Carroll, Tanmoy Sarkar, Akmaljon Salimov, Benjamin J. Kelly, Electra D. Paskett, Jesse J. Plascak, Shannon McWeeney, Jeffrey W. Tyner, Jeffery Klco, Nathan Salomonis, H. Leighton Grimes, Elaine R. Mardis, Ann-Kathrin Eisfeld. Novel Rho-GTPase regulatory protein gene family variants are frequent and associate with poor survival in patients (pts) with acute myeloid leukemia (AML) [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 7897.

Abstract Despite the success of immune checkpoint blockade (ICB), many patients fail to respond, highlighting a need for predictive biomarkers. We previously identified pre-treatment differences in peripheral blood mononuclear cells (PBMCs) from patients treated with ICB using an 11-color flow cytometry panel. These differences grouped patients into 3 distinct immune phenotypes, or immunotypes (Shen et al Sci Trans Med, 2021). Immunotype 1 (IT-1) was characterized by higher numbers of LAG-3+CD8+ T cells and associated with poor response and overall survival (OS) after anti-PD-1 therapy. IT-2 was characterized by lower LAG-3+CD8+ and better outcomes; IT-3, by the presence of additional cell populations not present in IT-1 or IT-2. Here, we aim to establish a deeper understanding of LAG-3+CD8+ T cells, the defining cell population of the IT-1 phenotype. We analyzed banked pre-treatment PBMC samples from patients with melanoma (n=30) and urothelial carcinoma (UC, n=48) previously assessed to have IT-1, IT-2, or IT-3, using a 28-color spectral flow cytometry assay, with single-cell RNA sequencing of a representative IT-1 sample. Compared with LAG-3-CD8+ cells, LAG-3+CD8+ T cells displayed higher expression of cytotoxic, terminal differentiation markers (GzmB, CD57, T-bet, Eomes) and lower expression of homing (CCR4, CXCR5, CCR7) and costimulatory molecules (ICOS, CD27, CD28, CD127). Consistent with our flow cytometry data, transcriptomic profiling revealed upregulation of GZMB, B3GAT1, TBX21, and EOMES, as well as downregulation of CCR4, CCR7, ICOS, CD27, CD28, and IL7R. These findings were further validated in an independent melanoma scRNA-seq dataset (Huuhtanen et al J. Clin. Invest, 2023). Finally, we evaluated samples from patients with the IT-2/3 phenotypes, confirming this pattern to be unique to IT-1. Our results reveal a distinct phenotypic signature defining LAG-3+CD8+ T cells, the hallmark of IT-1. These findings refine the definition of IT-1, highlight immune-phenotype differences between the IT-1 and IT-2/3, and suggest mechanisms, such as altered costimulation and trafficking, that underlie ICB resistance. Citation Format: Ariel Kogan Zajdman, Cory A. Brennick, Kayla J. Foster, Sydney A. Riddick, Tatiana Shcheglova, Matthew Adamow, Jasme Lee, Ronglai Shen, Katherine S. Panageas, Xiyu Peng, Margaret K. Callahan. Profiling of peripheral LAG-3+CD8+ T cells: An immune cell population associated with immunotherapy resistance [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 5243.