Mutant spliceosome proteins (e.g., U2AF1S34F, SF3B1K700E, or SRSF2P95H) alter RNA splicing in myeloid neoplasms, leading to increased production of nonsense transcripts. Inhibiting the nonsense-mediated RNA decay (NMD) pathway, which is responsible for degradation of nonsense transcripts, preferentially kills cells expressing mutant spliceosome proteins in vitro. In this study, we used an inhibitor of the kinase SMG1, a key regulator of NMD, to provide in vivo evidence that NMD is a therapeutic vulnerability for splicing factor mutant myeloid neoplasms. Primary mouse acute myeloid leukemia cells and human K562 leukemia cell lines expressing splicing factor mutants were more sensitive than wild-type cells to in vivo inhibition of SMG1 (SMG1i). Disruption of NMD activity by SMG1i led to increased R-loop levels in spliceosome wild-type cells, which were further increased in treated U2AF1S34F cells. This R-loop accumulation was accompanied by an increase in DNA damage. Degradation of R-loops with RNase H1 rescued spliceosome mutant cells from NMD inhibition-induced cell death. In U2AF1S34F cells, SMG1i increased NMD transcript isoforms (with reduced but detectable protein levels), which were enriched for DNA repair genes, including ATR and RAD51. Consequently, SMG1i-induced cell death in splicing factor mutant leukemias could be further enhanced by inhibition of ATR or RAD51. This study shows that in vivo targeting of NMD is a therapeutic strategy to treat myeloid neoplasms with aberrant splicing.

Copy number alterations (CNAs) accumulate non-randomly within cancer genomes reflecting specific DNA damage and repair events. Higher-order patterning of CNAs can illuminate the types and determinants of genome instability (GI), as well as their clinical relevance, highlighting the need to develop analytical frameworks to capture such patterns. To address this issue, we collated a literature-curated compendium of pre-defined CN-based GI scores and extracted de novo CN signatures. Application to 2,763 breast cancer genomes from The Cancer Genome Atlas and METABRIC revealed the complementarity of various GI scores and their differences across immunohistochemical subtypes. Of the eight CN signatures identified, three associated with distinct characteristics of homologous recombination deficiency and showed differential activity between cases with BRCA1 versus BRCA2 loss. Segments assigned to a HER2+ enriched signature strongly overlapped regions of chromothripsis and circular extrachromosomal DNA, suggesting that a common mutational process contributes to these phenotypes. CN "quiet" diploid and tetraploid genomes were apparent, with the latter group capturing a unique subset of whole genome doubled tumors enriched for PIK3CA, MAP3K1, and CDH1 mutations. Finally, combining CN signatures with tumor microenvironment analyses, patients with quiet genomes and low macrophage infiltration showed remarkably better survival outcomes. Collectively, these findings demonstrate the value of deep interrogation of scores and signatures in characterizing the biological processes and clinical implications underlying CN-based GI. The publicly available web portal (https://cnavisualizer.pittlabgenomics.com/home) will facilitate similar analyses across pan-cancer genomes.

Perturbations in DNA replication can impair fork stability, resulting in cumulative DNA replication stress. As activation of the DNA stress response elicits immunomodulatory effects, understanding the crosstalk between the epigenetic control of replication fork stability and the recruitment of immune cells may represent an actionable avenue to potentiate the sensitivity of tumors to immunotherapy. Here, we identified DPY30, a member of the histone methyltransferase WRAD/COMPASS complex, as a replication stress-specific epigenetic modifier in pancreatic ductal adenocarcinoma (PDAC). While other WRAD components broadly regulate transcription, DPY30 distinctively promoted H3K4me3 deposition at stressed DNA replication forks to safeguard DNA replication stability without altering global gene expression. Loss of DPY30 destabilized stalled forks causing fork degradation, chromosomal instability, and inflammation without reducing cancer cell proliferation. Consequently, T-cell infiltration induced by DPY30 deficiency promoted a tumor response to immune checkpoint blockade (ICB). In PDAC patients, high DPY30 tumor expression was associated with poorer ICB response, underscoring the potential of DPY30 as a predictive biomarker of immunotherapy response. Together, this study redefines our understanding of a replication stress-specific epigenetic code, unveiling DPY30 as a chromatin switch essential for stressed fork stability and a potential therapeutic target.

A major limitation in applying chimeric antigen receptor (CAR) T cells to solid tumors is toxicity in healthy tissues caused by a lack of tumor-specific targets. A promising strategy to overcome this deleterious cytotoxicity is to engineer control into CAR T cells beyond that conferred by antigen recognition alone. In a recent issue of Nature Chemical Biology, Scheller and colleagues report the development of a CAR that is inactivated through introducing the small molecule, venetoclax, which is a clinically approved targeted Bcl-2 inhibitor. The authors design venetoclax-dependent release of the CAR extracellular binding domain, thereby disrupting T cell contact with tumor cells and suppressing cytotoxicity. Furthermore, they demonstrate the reversibility of this approach, as withdrawal of the drug restores CAR T cell function. This work establishes a foundation for clinically translatable remote-controlled CAR T cell therapy for solid tumors.

The cellular response to hypoxia is driven by hypoxia-inducible factors (HIFs), which regulate genes involved in glycolysis, angiogenesis, and cell proliferation, as well as inflammation and tumor progression. HIF activation is well-characterized and is primarily regulated by oxygen-dependent prolyl hydroxylation and subsequent degradation. SET1B, a histone H3 lysine 4 (H3K4) methyltransferase, has recently emerged as a key modulator of HIF target gene transcription, but evidence suggests that it plays a broader role in modulating HIF transcriptional activity beyond histone methylation. Here, we revealed that SET1B interacts with RNA polymerase II to coordinate sustained HIF-mediated transcriptional activity through multiple functional domains. In clear cell renal cell carcinoma (ccRCC), SET1B was critical for sustained HIF activity, and SET1B expression correlated with disease progression and metastasis in patient samples. Moreover, SET1B depletion enhanced the efficacy of HIF-2 inhibitors. These findings establish SET1B as a driver of tumor progression and potential therapeutic target in ccRCC.

Maintaining sustained deoxyribonucleotide triphosphate (dNTP) pools is essential for DNA replication fidelity and genome stability. In EGFR-mutant non-small cell lung carcinoma (NSCLC), we found that disruption of dNTP homeostasis plays a critical role in determining sensitivity to the EGFR inhibitor osimertinib and in shaping mechanisms of acquired resistance. Transcriptomic and biochemical analyses revealed that osimertinib suppresses RRM2 expression, a key regulator of dNTP synthesis, through downregulation of the transcription factor MYBL2. In response to osimertinib-mediated replication stress and dNTP depletion, cells activated a compensatory pathway involving the stress-inducible ribonucleotide reductase subunit RRM2B via a transcriptional regulator, TNNT3. CHK2 signaling was essential for TNNT3 nuclear translocation and RRM2B transcriptional activation. Inhibition of CHK2 or combined CHK1/2 blockade impaired RRM2B induction, exacerbated replication stress, and delayed the development of osimertinib resistance both in cell lines and in xenograft models. Collectively, these findings reveal that EGFR-mutant NSCLC cells rely on dynamic signaling through EGFR-MYBL2-RRM2 and CHK2-TNNT3-RRM2B regulatory pathways to maintain dNTP pool balance under therapeutic pressure. Disruption of this signaling network sensitizes tumors to osimertinib and impairs the acquisition of resistance, linking metabolic regulation to therapeutic resistance and disease progression.

Abstract Introduction: Replication repair deficiency (RRD) is a pan-cancer mechanism caused by germline and/or somatically acquired mutations in the replication repair machinery - DNA polymerase proofreading and the mismatch repair (MMR) system. Germline monoallelic (Lynch Syndrome, LS) or biallelic (Constitutional Mismatch Repair Deficiency, CMMRD) mutations in MMR genes are present in 5-10% of glioblastomas in children, adolescents, and young adults. RRD gliomas are lethal, chemoradiation-resistant cancers, characterized by universal hypermutation and variable susceptibility to immune-checkpoint inhibition (ICI). These tumors exhibit variability in patient age of onset, type, location, and response to ICI. Methods: To understand the clinical and biological differences associated with RRD central nervous system (CNS) tumors, we used germline mutations and brain development-specific Cre-drivers to generate murine models that recapitulate the phenotypic and genomic characteristics of each human RRD subgroup: 1) MMRD+PPD (Nestin- and Olig2-Cre+/ Msh2LoxP/LoxP/PoleS459F/+ and LSL-PoleP286R/+): MMR-deficiency (MMRD) in combination with polymerase proofreading deficiency (PPD). 2) MMRD-only (Nestin-Cre+/Trp53LoxP/LoxP and Msh2LoxP/LoxP or Mlh1-/-): MMRD lacking PPD associated with TP53 mutations. Results: Using trans-species comparative approach, we elucidated a mechanistic model of RRD-driven brain tumorigenesis. We revealed that the cell-of-origin significantly contributes to determining brain tumor type, location, and age of tumor onset, suggesting a strong impact of early- or late-RRD mutational onset in shaping tumor biology (p<0.0001). Importantly, using murine neural stem cells, we discovered that germline mutagenesis onset directly influences timeline of brain tumor formation and survival between CMMRD and LS patients (p<0.05). We further demonstrate the interplay between POLE mutations and MMRD status in modulating the likelihood of brain tumorigenesis in both species. To understand the interaction between hypermutation and the immune system, we characterized the tumor immune microenvironment in spontaneously forming tumors. We uncovered subgroup-specific immune landscapes, with CD8+ T cell activity emerging as a key modulator in controlling brain tumor growth (p<0.0001), suggesting an underlying mechanism that may inform therapeutic strategies in RRD patients. Significance: Altogether, our models accurately mimic the human condition, providing a mechanistic framework of RRD-driven brain tumorigenesis, optimization of subgroup-tailored immunotherapy approaches, and putative surveillance protocols. Citation Format: Zoya Aamir, Melissa A. Galati, Emma Gattoni, Owen Crump, Nemanja Ilic, Anirban Das, Nicholas R. Fernandez, Angel K. Wong, Lucie Stengs, Jose R. Dimayacyac, Yuan Chang, Vanessa Bianchi, Melissa Edwards, David Malkin, Cynthia Hawkins, Nuno M. Nunes, Uri Tabori. Trans-species analysis of central nervous system developmental-specific replication repair deficiency reveals differential patterns of gliomagenesis and response to immunotherapy [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 631.

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: Spatial imaging outputs continue to grow in scale and complexity. While brightfield IHC and H&E remain the qualitative gold standard for antibody-based assessment, mIF offers quantitative protein measurement on a single slide. However, challenges such as non-specific binding, imaging artifacts, and variability across sites and operators limit confidence in mIF reproducibility. A quantitative, robust method is needed to assess concordance between IHC and mIF stains. Methods: Using a pan-cancer dataset with a 4-plex mIF panel and matched IHC sections from consecutive slides, we first co-registered images into a shared coordinate space with Valis, applying global rigid and non-rigid transformations from feature matches. IHC stain channels were isolated via stain-matrix-based deconvolution. A tissue mask was generated on the mIF image using Otsu thresholding and morphology operations and then projected onto the IHC slide.Tissue was divided into tiles whose size accounted for section-to-section distance, registration error, and biological variability. Within each tile, random windows were sampled to perform two tests: (1) identify whether the tile contains high stain intensity and (2) determine whether the corresponding IHC and mIF tiles exhibit statistically concordant staining. This approach yields both a DICE score for high-stain region overlap and a stain concordance metric capturing agreement across high- and low-stain regions. Tile-level results and heatmaps are visualized in SpaceIQ™. Results: Concordance between mIF and IHC varied substantially across markers, with CD8 showing the highest and FoxP3 the lowest agreement, a trend consistent across samples. Concordance heatmaps also revealed strong spatial effects, with some tissue regions highly concordant and others clearly discordant. Expert visual review matched these quantitative findings. Conclusions: This segmentation-free framework identifies substantial marker- and region-specific variation in concordance between mIF and IHC staining. Because the method is marker-agnostic and compensates for registration error and inter-section biological differences, it provides a generalized, quantitative approach for evaluating agreement between paired mIF and IHC slides across platforms. Citation Format: Brian Falkenstein, Raymond Yan, A. Burak Tosun, S. Chakra Chennubhotla, Filippo Pullara. Robust segmentation-free stain quality concordance metrics in the SpaceIQ™ multi-omic analysis platform [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 715.

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.