Citations

Abstract Introduction: Long intergenic noncoding RNAs (lincRNAs) and circular (circ) RNAs have been shown to play key roles in carcinogenesis and tumor progression. Urine represents a promising source of tumor-reflective signals shed directly from the urothelial microenvironment in bladder cancer (BC) patients. Here, we evaluate the urine cell-free (cf) RNA transcriptome in a cohort of 50 patients with localized BC. Methods: A total of 50 localized BC patients and 24 healthy adults were enrolled. Pre-operative urine (collected on the day of radical cystectomy) was processed to isolate cfRNA from urine supernatant. Libraries were prepared using an Illumina tagmentation-based RNA enrichment workflow and sequenced to 100M paired-end reads per sample on a NovaSeq X. FASTQ files were concatenated per sample and QC-checked with RNA-SeQC and Samtools. Reads were aligned to GRCh38/hg38 with STAR, and gene counts were generated using Feature Counts. Linear differential expression analysis (DEA) was performed with DESeq2. For discovery we used random halves of TCGA-BLCA and healthy urine cfRNA samples, alongside a second DEA with random halves of urine BC cfRNA vs. controls; overlapping upregulated genes were validated in the remaining samples. For circular (circ) RNAs, annotations from MiOncoCirc were used; junctions with ≥5 supporting reads were retained, and DEA was conducted with edgeR. Results: In this study, the median age was 68 years, and 80% (40/50) were male. Among the cohort, 66% (33/50) had muscle-invasive bladder cancer (MIBC), of whom 54% received neoadjuvant chemotherapy. Differential expression analysis of TCGA-BLCA tumors and BC urine cfRNA, identified 27 genes commonly upregulated, with 23 of the 27 shared genes strikingly being long intergenic noncoding RNAs (lincRNAs). These BC/urine-specific lincRNAs—including LINC00930, LINC01004, DLG5-AS1, APTR, PSORS1C3, and SERPINB9P1—have known roles in proliferation, EMT, metabolism, and immune signaling. Outside of lincRNAs, tumor-reflective coding transcripts in BC/urine were enriched for autophagy, driven by MAP1LC3B2.TCGA-BLCA survival analysis showed that LINC00930 and PSORS1C3 were associated with worse overall survival. Expanding beyond linear RNA sequences, analysis of urine circRNAs from BC patients revealed a strong cancer-specific dysregulation pattern, with 77 circRNAs significantly upregulated compared with healthy controls. Upregulated urine circRNAs arose from host genes involved in BC-relevant pathways, including invasion (DOCK1, ARHGAP5), autophagy/ER stress (VMP1, ERN1), and luminal differentiation (GRHL2, ESRP1). Conclusion: Cell-free RNA profiling reveals a distinct transcriptomic signature including differential lincRNA- and circRNA-driven autophagy, EMT, proliferation, metabolic, differentiation, and immune signaling programs in the urine of bladder cancer patients. Citation Format: Pradeep Chauhan, Li Lin, Irfan Alahi, Jessica Linford, Lilli J. Greiner, Ayesha Hashmi, Anushka Viswanathan, Nathan Colon, Faridi Qaium, Peter K. Harris, John Sheng, Jacob J. Orme, Eric H. Kim, Zachary L. Smith, Woodson Smelser, Christopher A. Maher, Aadel A. Chaudhuri. Urine cell-free RNA captures tumor-reflective long intergenic noncoding and circular RNA programs in bladder 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 1133.

Abstract Background: Endometrial cancer (EC) remains a major gynecologic malignancy driven in part by aberrant estrogen receptor (ER) signaling. The active tamoxifen metabolite (Z)-endoxifen exhibits dual SERM/SERD-like activity, modulating ER function while exerting additional noncanonical antiproliferative effects. Understanding (Z)-endoxifen’s mechanisms of action and its interaction with cell-cycle regulators is critical to optimizing targeted therapy in EC. This study investigated the molecular and functional effects of (Z)-endoxifen, alone and in combination with the CDK4/6 inhibitor abemaciclib, across diverse EC models to define mechanistic activity and translational potential. Methods: ER+ (Ishikawa, HCI-EC23, patient derived organoid U1561.005) and ER-variable (ARK1, ARK2) EC models were treated with estrogen (0-10 nM), (Z)-endoxifen (125 nM-10 µM) or fulvestrant (0-2.5 μM), and abemaciclib (0-10 μM), as single agents or in combination. Cell viability was assessed using luminescence-based assays. Dose-response analyses and combinatorial synergy were evaluated to determine endocrine sensitivity, ER dependency, and downstream cell-cycle effects. Results: Estrogen induced biphasic proliferative responses in ER+ models, confirming ligand-driven mitogenic signaling. (Z)-Endoxifen monotherapy suppressed viability across all cell lines, demonstrating superior potency to fulvestrant and activity independent of ER expression status. Mechanistically, (Z)-endoxifen abrogated estrogen-induced proliferative peaks, consistent with ER antagonism and potential receptor degradation, while also exerting ER-independent effects suggestive of cell-cycle modulation. Abemaciclib monotherapy inhibited viability in both ER+ and ER− models, and its combination with (Z)-endoxifen yielded strong synergistic activity (Chou-Talalay combination index < 1), including in ER-negative ARK2 and the ER+ U1561.005 organoid. This synergy likely arises from concurrent disruption of ER signaling and CDK4/6-cyclin D regulatory pathways. Conclusions: (Z)-Endoxifen demonstrates dual ER-dependent and ER-independent activity in EC preclinical models. Synergy was observed in combination a CDK 4/6 inhibitor. These findings broaden the current understanding of (Z)-endoxifen’s efficacy and support further exploration of its potential therapeutic role in ER-driven and mixed-phenotype EC, including evaluation of responses across ER expression levels. Citation Format: Grace Choong, Xiaonan Hou, Sandra Suarez Hammer, Scott M. Blackburn, John Weroha, Steven C. Quay. (Z)-endoxifen modulates estrogen receptor and cell-cycle signaling to induce synergistic antiproliferative effects with CDK4/6 inhibition in endometrial cancer 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 405.

Abstract Introduction: Ewing sarcoma (EwS) is a FET::ETS family member-driven primary bone cancer demonstrating vast heterogeneity. Between a patient’s primary diagnosis and disease progression, tumor cell state adaptation, microenvironment changes, and the landscape of evolving therapeutic vulnerabilities remain poorly understood. To address these gaps, the Sean Karl Cohort was established in 2025 to conduct the largest single-cell transcriptomic analysis of retrospective paired tumor samples from patients with EwS. Here, we present data from the five analytic teams, and through shared SOPs now expand this cohort to Europe. Methods: Common sample processing SOPs were established to isolate cells from FFPE material from paired patient EwS samples. Single-cell (sc) RNAseq was generated using the GEM-X Flex Gene Expression protocol (10x Genomics). Alex’s Lemonade Stand Foundation (ALSF) Data Lab established a common data processing and integration pipeline with an EwS-specific cell annotation workflow to create a harmonized dataset for downstream analyses. Analytic teams were created to define tumor cell subpopulations and their therapeutic vulnerabilities and to characterize the tumor microenvironment. Data: We demonstrate the feasibility of generating high-quality scRNAseq profiles from retrospective FFPE-preserved EwS tumors. To date, 116 samples have yielded ∼800,000 cells. Integrated analysis reveals reproducible EwS tumor cell states shared across patients with regulatory network analyses nominating candidate therapeutic vulnerabilities. The scale of this international cohort enables saturation analysis for rare cell populations. Paired and longitudinal samples further allow correlation of emergent cell states with therapy resistance and metastatic progression. For example, immune-focused analyses show increases in T cell and macrophage populations in post-therapy samples. A novel, standardized EwS-specific cell annotation workflow has been developed to harmonize analyses and findings. These data will be openly shared with the community through the ALSF Single-cell Pediatric Cancer Atlas Portal. Conclusion: Large-scale international collaborative sample sharing, standardized processing pipelines, and harmonized EwS-specific annotations now enable deep single-cell analyses to characterize tumor heterogeneity in this rare pediatric and adolescent cancer. An in vivo expansion of the Sean Karl Cohort is underway to assess conservation of EwS tumor cell states and regulatory vulnerabilities identified in human tumors pre- and post-therapy in preclinical models, further supporting translation of these findings toward therapeutic strategies. Citation Format: Abbe Pannucci, Elina Mukherjee, Jessica D. Daley, Shireen Sita Ganapathi, Elissa Boguslawski, Lea Surrey, Lauren Gutstein, Patrick Azar, Emily Stockfisch, Azfar Neyaz, Ivy John, Jennifer Picarsic, Yutaro Tanaka, Byron Butaney, Riaz Gillani, Brian D. Crompton, Katherine A. Janeway, Jaclyln Taroni, Jessica Davis, Damon Reed, Adam Shlien, Theodore W. Laetsch, Rajen Mody, Clémence Henon, Thomas G. Grünewald, Elizabeth R. Lawlor, Filemon Dela Cruz, Patrick J. Grohar, Jovana Pavisic, Ally Hawkins, Anthony R. Cillo, Kelly M. Bailey. The Sean Karl Cohort: An international single-cell RNAseq study of paired patient Ewing sarcoma specimens [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 643.

Abstract Breast cancer is the most common female malignancy in the U.S. and the leading cause of cancer-related deaths in women worldwide. Nearly 80% of breast tumors express estrogen receptor alpha (ERα) which drives tumor progression. While endocrine therapies and CDK4/6 inhibitors have increased overall survival, many patients experience recurrence of metastatic disease that is largely refractory to additional lines of endocrine therapy. Metastatic disease remains incurable, highlighting the need for alternative treatment approaches. Through a genome-wide CRISPR knockout screen, we identified Lemur Tail Kinase 2 (LMTK2) as the top hit exhibiting synthetic lethality in the presence of the endocrine therapy, endoxifen, in both endocrine-sensitive and -resistant cell line models. Using publicly available patient tumor databases we identified LMTK2 copy number gain/amplification in 67% of ERα+ human metastatic breast tumors, while only 1% of primary tumors harbored these genomic alterations. Elevated LMTK2 expression in early-stage primary breast tumors was associated with worse recurrence-free survival and overall survival. We discovered that LMTK2 mRNA and protein levels were substantially elevated in nearly all cell-line and patient-derived models of endocrine and CDK4/6i resistance. Utilizing siRNAs and dox-inducible shRNA expressing cell lines, we found that knockdown of LMTK2 enhanced the efficacy of multiple endocrine therapies in treatment naïve models and resensitized resistant models to their respective treatment. LMTK2 overexpression was found to be sufficient for conferring resistance to all standard of care therapies tested including endocrine agents, CDK4/6i, and PI3K inhibitors. RNAseq and global proteomics of LMTK2 overexpressing and knockout models revealed the PI3K/AKT pathway and estrogen signaling as highly regulated pathways by LMTK2. Confirmatory western blot studies demonstrated that LMTK2 overexpression results in dramatic increases in AKT and ERα phosphorylation and activity, while knockout of LMTK2 diminishes these effects. Attempts to completely ablate LMTK2 in endocrine/CDK4/6i-resistant models via CRISPR were not successful, and continuous siRNA or shRNA-mediated knockdown was lethal in resistant cell lines, confirming its essentiality in this setting. In summary we have uncovered LMTK2 as a novel mediator of endocrine/CDK4/6i resistance that is mechanistically linked to the PI3K/AKT pathway among others. Genetic manipulation of LMTK2 has validated it as an ideal therapeutic target for both advanced and newly diagnosed forms of ERα+ breast cancer. Citation Format: Anna Detry, Sofia Marigliano, Jenna Heinen, Adam Bass, Calley Jones, Rajeev Muthyala, Matthew Goetz, John Hawse. LMTK2: A novel driver of endocrine resistance in ERα+ 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 443.

Abstract Introduction: Attending to survivorship including distress in the contexts of social drivers (SDH) is critical but lacking. In response, this novel research on Black CaP survivors (CaPS) aims to measure SDH distresses and assess utility of a digital intervention to inform survivorship studies, practice and outcomes. Method: Informed by the Science of Survivorship, and our pilot study with multisectoral stakeholders, we assessed SDH and acceptability/utility of a digital Survivorship Care (SC) Toolkit. SCToolkit is a digitized whole-person care with guidance from multisectoral stakeholders e.g., clinicians and survivors. SCToolkit includes: ASCO informed treatment summary template for diagnostics, treatments, side effects, care team, genetics, and comorbidities, and patient active guide on survivorship concerns, provider communication, health advisories, survivorship care booklet, SDH FINDHELP resource, and survivorship videos. SCToolkit was delivered via survivors’ personal REDCap file, and can be uploaded to their EMR patient portal for easy-to-use survivorship roadmap accessible on smartphones, laptops, and computers. Motivational Interviewing was provided via Zoom to facilitate digital efficacy and SCToolkit use. Descriptive analyses were conducted using SAS. Citation Format: Kimlin Ashing, Floyd Willis, Christopher Williams, John McCall, Gaole Song, Douglas Adu-Fokuo, Erika Galdamez, Fornati Bedell, Cassandra N. Moore, EWAN COBRAN, Che Ngufor, Kathleen J. Yost, Timethia J. Bonner, Getachew Dagne, Arnold Merriweather, Ernest T. Kaninjing, Rotimi Oladapo, Roxana S. Dronca, Folakemi T. Odedina. Examining contextual distress and utility of a digital survivorship care toolkit [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 880.

Study Design: Retrospective analyses of the center of rotation (COR) in asymptomatic volunteers and symptomatic patients. Objective: Document (i) level-specific COR position at radiographically normal levels in asymptomatic volunteers, (ii) preoperative COR abnormalities in symptomatic patients, and (iii) correlations between COR and other intervertebral motion metrics. Summary of Background Data: The COR has been discussed in many prior publications; however, our understanding of the normal position of the COR, the types and prevalence of COR abnormalities in patients, and the association between COR and other more common measures of intervertebral motion remains limited. Methods: Using automated, FDA-cleared software, we quantified COR and standard motion/alignment metrics from cervical flexion-extension radiographs of 341 asymptomatic volunteers and 1263 symptomatic preoperative patients. Level-specific 95% reference ellipses were derived from volunteers. Abnormality was defined as COR outside the level-matched ellipse. Associations between COR (cranial-caudal and anteroposterior) and rotation, translation, sagittal plane offset, disc height change, and standardized instability indices were evaluated. Results: In volunteers, COR was level-dependent and well summarized by 95% reference ellipses. The cranial-caudal position of COR was chiefly determined by rotation and translation, whereas the anteroposterior position related to sagittal plane offset and disc height change. COR also correlated with translational (TI-Index) and vertical (AVI/PVI) instability metrics. In symptomatic patients, most levels fell within reference ellipses; when deviations occurred, they were infrequent and multidirectional. Conclusions: COR can be automatically derived from flexion-extension radiographs. Deviations from level-specific reference ellipses provide an intuitive visualization of abnormal kinematics, complementing established motion metrics and linking cranial-caudal and anteroposterior shifts to specific patterns of translation, alignment, and instability.

ABSTRACT BACKGROUND Increasing evidence suggests that accurate prediction of Alzheimer’s disease (AD) symptom onset requires more than amyloid- and tau-centric biomarkers such as cerebrospinal fluid (CSF) Aβ42/40, total tau and p-tau181 and plasma p-tau217. Autosomal dominant AD (ADAD), caused by pathogenic PSEN1, PSEN2 and APP mutations with predictable age at symptom onset, presents a unique opportunity to characterize the chronological changes in proteins beyond amyloid and tau and clarify them as early biomarkers of disease onset or as biomarkers related to disease staging and progression monitoring. METHODS We measured 972 CSF samples corresponding to 484 participants of the Dominantly Inherited Alzheimer Disease Network (DIAN) using the NULISASeq 120 CNS Disease Panel. We first benchmarked the technology against gold-standard measurements followed by the identification of proteins that were differentially abundant in relation to mutation status and symptomatology. Next, we determined the chronological emergence of protein changes in relation to the estimated years to onset (EYO). Finally, we assessed whether specific protein measures improved the prediction of EYO in the ADAD. FINDINGS NULISA measurements were comparable to those previously published. We demonstrated that known early alterations in CSF amyloid and tau were followed by inflammatory and neurodegenerative responses suggesting that clinical manifestation of AD happens before the inflammatory processes is fully developed. Finally, we found a multi-protein composite approach for predicting EYO that outperformed single biomarker values. INTERPRETATION Our results suggest that the main CSF proteomic landscape changes in ADAD are due to the presence of a pathogenic mutation and occur prior to symptom onset. Improved performance of multi-protein composite to predict EYO compared to single biomarker values highlights the added value of multiplex proteomic signatures for biomarker panel development. FUNDING National Institute on Aging, Alzheimer’s Association, German Center for Neurodegenerative Diseases, Raul Carrea Institute for Neurological Research, Japan Agency for Medical Research and Development, Ministry of Health & Welfare and Ministry of Science and ICT, Republic of Korea, Spanish Institute of Health Carlos III.