Figure S6. Differential pathway gene set enrichment analysis among DKOAA vs DKO in all cell types, visualized as networks via aPEAR.

Histone modifications, including Nε-lysine acetylation and methylation, play critical roles in the regulation of eukaryotic transcription. The addition of acetyl and methyl groups and removal of acetyl groups to histones involve redox-neutral reactions. Demethylation is O2-dependent, as reported for reactions catalysed by the 2-oxoglutarate-dependent hypoxia-inducible factor (HIF) hydroxylases, one of which is structurally related to the Jumonji-C (JmjC) histone demethylases. We screened for substrates of the HIF-regulated JmjC lysine demethylase KDM3A and unexpectedly observed that purified recombinant KDM3A catalyses oxidation of the Nε-acetyl group of the Lys-9 of histone H3 (H3K9ac) giving an Nε-hydroxyacetylated product (H3K9acOH). Here we show that Nε-hydroxyacetyl-lysine is recognized by proteins known to bind to H3K9ac, including histone deacetylases and the YEATS domain-containing AF9. Studies employing an Nε-hydroxyacetyl-lysine selective antibody and mass spectrometry support the cellular relevance of Nε-hydroxyacetyl-lysine. Our combined biochemical and cellular results provide evidence for an unanticipated O2-mediated link between histone lysine Nε-acetylation and JmjC catalysis.

Amyloidosis comprises a heterogeneous group of disorders marked by the extracellular deposition of insoluble protein fibrils, known as amyloids, which can disrupt normal tissue architecture and lead to organ dysfunction. Accurate identification and subtyping of the amyloidogenic protein are critical for clinical management as treatment strategies vary significantly depending on the underlying protein species. While conventional diagnostic tools such as Congo red staining and immunohistochemistry are commonly used, they suffer from limited specificity and antibody availability, often resulting in misclassification. Mass spectrometry (MS)-based proteomics has become the gold standard for amyloid subtyping, offering unmatched sensitivity and proteome-wide coverage.Here, we describe a spatial proteomics protocol that integrates laser capture microdissection (LMD) with advanced LC-MS/MS acquisition methods, including data-dependent acquisition (DDA), data-independent acquisition (DIA), and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS). With the integration of these methods, we present the analysis of formalin-fixed paraffin-embedded (FFPE) tissue sections. This comprehensive workflow enables precise excision of amyloid-rich regions and enhances detection of amyloid proteins and co-deposited biomarkers from minimal tissue input. The combination of FAIMS with DIA and DDA not only improves the depth of proteomic coverage but also increases reproducibility and sensitivity, making it particularly suitable for low-abundance samples. This protocol provides a robust and scalable platform for the accurate molecular subtyping of amyloidosis and has the potential to inform personalized therapeutic decisions in clinical pathology.

Abstract Pancreatic cancer (PDAC) cells experience nutrient starvation in a poorly perfused tumor microenvironment. Metabolic dependencies that protect PDAC cells from detrimental oxidative stress in a nutrient-restricted niche represent as tumor-specific targets. While the role of mitochondria in supporting energy production and biosynthetic requirements of cells has been well investigated, their contribution to maintaining intracellular redox homeostasis when PDAC cells are exposed to nutrient deprivation is unknown. Our results demonstrate that cytosolic transport of citrate via SLC25A1 confers a survival advantage to PDAC cells by protecting them from ferroptosis, a well-established iron-dependent cell death mechanism, under nutrient-limited conditions. Employing selective SLC25A1 inhibitor or targeting mitochondrial OXPHOS dramatically reduced GPX4 expression and PDAC cell viability. Rescuing GPX4 expression with the products of both ACLY and ACO1-dependent pathways uncovered their critical role in conferring survival advantage under metabolic stress. Importantly, exogenous expression of GPX4 reversed redox imbalance and metabolic discordance resulting from the lack of SLC25A1 activity, indicating the requirement of citrate-induced GPX4 expression to support mitochondrial health and function. As observed with cultured cells under nutrient limitation, SLC25A1 function was revealed to be indispensable in pancreatic tumor microenvironment, and the reduced growth, due to the lack of SLC25A1 activity, was rescued with antioxidant NAC in preclinical models of PDAC. Lastly, SLC25A1 suppression was accompanied by elevated glutamine metabolism, and combination therapy with pharmacologic inhibitors of SLC25A1 and glutaminase inhibitor CB-839 dramatically suppressed tumor growth, highlighting this combinatorial approach as a potential therapeutic strategy in PDAC. Citation Format: Adam Kneebone, Kailey Lindaur, Ata Abbas, Joel Cassel, Sarah Graff, Caudia Rose Keating, Gerard Abood, Xianzhong Ding, William Small, Clodia Osipo, Wei Qiu, Curtis Tatsuoka, Simone Sidoli, Costas Andreas Lyssiotis, Joseph M. Salvino, Ali Vaziri-Gohar. Cytosolic transport of citrate protects nutrient-austere pancreatic cancer from ferroptosis [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 7325.

Abstract DNA hypomethylating agent (DNMTi) efficacy is associated with the re-expression of epigenetically silenced tumor suppressor genes (TSGs) and transposable elements (TEs) in preclinical cancer models, yet their clinical efficacy in solid tumors is limited. An emerging mechanism of resistance to DNMTi involves compensation through repressive histone post-translational modifications (PTMs). Here, we define a targetable chromatin-based mechanism through UHRF1-SUV39H1/H2 crosstalk that reinforces transcriptional silencing in colon cancer cells exposed to DNMTi’s. Leveraging integrative epigenomic profiling and biochemical analyses, we discovered that transient DNA hypomethylation triggers UHRF1-dependent mono-ubiquitination of lysine 18 on histone H3 (H3K18ub), which in turn stimulates SUV39H1/H2 methyltransferases to deposit H3K9me3 at CpG island promoters of DNA methylation-silenced TSGs. This UHRF1-SUV39H1/H2 crosstalk establishes new heterochromatin domains that stabilize repression despite global DNA demethylation. Disrupting UHRF1 enzymatic function or an identified H3K18ub-recognition motif in SUV39H1 prevents H3K9me3 accumulation, increases TSG re-expression, and enhances DNMTi-induced antiproliferative effects in colon cancer cells. These findings reveal a compensatory heterochromatin signaling mechanism that limits DNMTi responses and identify the UHRF1-SUV39H1 pathway as a novel therapeutic target to improve epigenetic therapy efficacy in solid tumors. Citation Format: Yanqing Liu, Joel A. Hrit, Alison A. Chomiak, Stephanie Stransky, Jordan Hoffman, Rochelle L. Tiedemann, Ashley K. Wiseman, Leena S. Kariapper, Bradley M. Dickson, Evan J. Worden, Christopher J. Fry, Simone Sidoli, Scott B. Rothbart. Targeting UHRF1-SUV39H1/H2 crosstalk enhances DNMT1 inhibitor efficacy in colon 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 1958.

Table S9. Compositional analysis (differential abundance analysis) comparing proportions of cell subtypes from each genotype using the Propeller test from the Speckle package.

SUMOylation is a dynamically regulated post-translational modification involving covalent attachment of small ubiquitin-like modifiers (SUMOs) to lysine residues of target proteins. SUMOylation modulates multiple fundamental host cellular pathways, including pathways hijacked by HIV-1 to enable replication, but has not been explored by large-scale proteomics in the context of HIV-1 infection. Here, we performed a proteome-wide, mass spectrometry-based screen to identify proteins that are SUMOylated in response to HIV-1 infection. We show that infection with HIV-1 leads to the widespread increased SUMOylation of the heterogeneous nuclear ribonucleoprotein (HNRNP) A/B family. This phenotype was driven by expression of HIV-1 Viral Infectivity Factor (Vif), suggesting an unexplored function for this protein. Depletion of HNRNP A/B proteins led to altered splicing of HIV-1 viral RNAs and dramatically reduced HIV-1 infectivity. Our data suggest a mechanism involving HIV-1-induced, Vif-mediated SUMOylation of host RNA splicing factors as a means to regulate HIV-1 alternative splicing.

Figure S20. DE across OS models, stratified by pathologic subtype for which samples were available (including Osteo and Fibro-like, but not chondro-like).

Summary DNA replication initiation in higher eukaryotes occurs at thousands of sites distributed throughout the genome and follows a defined temporal program. However, replication origins are not determined by a conserved DNA motif, and how specific genomic regions are selected for initiation remains poorly understood. Here, we identify a transcription-associated histone modification in human cells, mono-methylation of histone H3 lysine 37 (H3K37me1), and show that it regulates the spatial organization of replication initiation. H3K37me1 is enriched across actively transcribed gene bodies, and its depletion leads to a redistribution of replication origin activity toward intragenic regions. We show that H3K37me1 limits the association of the MCM2–7 replicative helicase with transcribed chromatin, thereby restricting intragenic origin usage. Consistently, loss of H3K37me1 increases both MCM2–7 occupancy and replication initiation across gene bodies. Collectively, our findings uncover a chromatin-based mechanism that couples transcription with DNA replication origin selection by limiting unscheduled origin firing within transcribed regions.

Abstract Acquired and intrinsic resistance to targeted and immune therapies in melanoma is associated with an undifferentiated, mesenchymal-like tumor cell state characterized by low expression of the melanocytic lineage regulator MITF and high expression of the receptor tyrosine kinase AXL. Because MITF governs lineage-specific survival and differentiation programs, therapy-resistant MITF-low/AXL-high melanomas appear to evade these programs and instead depend on alternative survival mechanisms. To uncover such dependencies, we systematically interrogated genome-scale CRISPR-Cas9 knockout screens from the Cancer Dependency Map and identified a selective vulnerability to the epigenetic regulator lysine-specific demethylase 1 (LSD1/KDM1A). Intriguingly, domain-focused CRISPR screening spanning whole LSD1 coding locus further revealed that this dependency is mediated through non-catalytic, scaffold-like functions of LSD1 rather than its demethylase activity. Mechanistically, integrated LSD1 ChIP-seq, histone ChIP-seq (H3K4me1/2/3 and H3K27ac) and RNA-seq analyses demonstrated that LSD1 sustains a survival program characterized by chromosome segregation and neuronal dedifferentiation in MITF-low melanoma, orchestrated through distinct super-enhancer activation at key regulators, such as CDC6, related to these pathways. Disruption of LSD1’s non-catalytic activity by a LSD1 allosteric inhibitor induced G2/M arrest, transcriptional reprogramming, and growth inhibition selectively in MITF-low melanomas via downregulation of CDC6. Strikingly, genetic and pharmacological ablation of LSD1’s non catalytic function significantly prevented or delayed the emergence of resistance to BRAF inhibition and anti-PD-1 therapy in vitro, in vivo, and in patient-derived melanoma organoids. Furthermore, pan-cancer analyses revealed a similar LSD1 dependency in other poorly differentiated malignancies, including pancreatic cancer, that share an undifferentiated transcriptional state. Collectively, these findings define an epigenetically determined, therapeutically targetable state that underlies treatment resistance across multiple cancer types. By targeting the non-catalytic scaffolding function of LSD1, either alone or in combination with targeted and immune therapies, it may be possible to eradicate undifferentiated, drug-tolerant tumor cell populations and overcome therapeutic resistance in melanoma and other aggressive cancers. Citation Format: Shinichiro Kato, Anlun Xu, Hongyan Xie, Simone Sidoli, Genevieve Boland, Russel W. Jenkins, Dave S. Hoon, Hiroyoshi Nishikawa, David E. Fisher. Druggable dependency on histone demethylase LSD1 in MITF-low therapy resistant 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 1936.

Microsporidia such as Encephalitozoon hellem are obligate intracellular human parasites that remain genetically intractable, limiting functional characterization of their proteomes. Structural studies based on homology-based modeling and the use of deep learning algorithms of microsporidian proteins also remain limited because most have little to no sequence similarity to proteins with solved structures. To address these limitations, we developed an approach that incorporates cross-linking mass spectrometry (XL-MS) data into structure prediction. XL-MS data provides upper bound distance constraints that can be incorporated into protein deep-learning based modeling and subsequent docking. Using this approach, we generated a model for two interacting E. hellem spore wall proteins Spore Wall Protein 1B (Swp1b) and Endospore Protein 1 (EnP1), with no clear homologs outside of microsporidia, and which contain several disordered regions. These proteins are extremely abundant spore wall proteins of microsporidia and previously were not known to interact with one another. The resulting model not only is consistent with the experimental crosslinks used to generate the model but was subsequently confirmed by independently generated XL-MS data. The described AlphaLink-Modeller framework for structure prediction is particularly well suited to proteins with limited homology and/or substantial flexible regions, given they adopt a defined structural state within a biological context, thereby extending integrative modeling approaches to previously inaccessible targets.

The DNA hypomethylating agents (HMAs) 5-azacitidine and decitabine are the backbone of disease modifying therapy in myelodysplastic syndromes (MDS). Resistance to therapy often develops by upregulation of de novo pyrimidine synthesis, which competes directly with the DNMT1-depleting nucleotide Aza-dCTP by building natural cytidines and deoxycytidines from amino acid building blocks. Inhibition of de novo pyrimidine synthesis can restore sensitivity of leukemic stem cells to therapy. Here we demonstrate that pyrimethamine (PYR), a Food and Drug Administration-approved antiparasitic, antifolate agent causes apoptosis in multiple leukemic cell lines, has synergy with venetoclax (Ven) and additive effect with HMA's in HMA and Ven-resistant leukemic cell lines as well as murine models. It increases differentiation of stem and progenitor populations in primary samples of patients with MDS and is able to directly inhibit de novo pyrimidine synthesis thereby overcoming one of the primary mechanisms of HMA resistance. In combination with Ven, it also downregulates multiple enzymes implicated in pyrimidine synthesis.

Summary Sex differences strongly influence susceptibility to metabolic dysfunction-associated steatotic liver disease (MASLD), yet the regulatory mechanisms underlying these differences remain incompletely understood. To examine sex-specific hepatic adaptation to a high-fat (HF) diet mouse model of MASLD, we integrated proteomics, transcriptomics, and Oxford Nanopore direct RNA sequencing for transcriptome-wide m6A profiling in male and female mouse livers. Female mice were relatively protected from HF diet–induced hepatic steatosis and exhibited distinct proteome remodeling enriched for peroxisomal pathways. In contrast, transcriptomic responses in females were dominated by inflammatory signatures and did not recapitulate the metabolic adaptations observed at the protein level, revealing extensive RNA–protein discordance and post-transcriptional remodeling. Integrated RNA–protein analyses identified female-specific amplification of peroxisomal proteins despite modest transcript-level changes. HF diet also induced sex-specific remodeling of m6A RNA methylation and altered regulation of the m6A methylation system. Notably, reduced 3′ UTR m6A methylation of peroxisomal transcripts inversely correlated with increased protein abundance relative to RNA expression in female mice. Together, these findings implicate m6A-associated post-transcriptional regulation in sex-specific hepatic adaptation to HF diet exposure and the basis for discordance between many of the mRNAs and proteins in the liver.

Table S8. Marker genes for all celltype subclusters.

Figure S1. Clustering and markers of transgenic OS tumors.

Aging somatic cells are characterized by specific chromosome aneuploidy, particularly involving chromosome Y (ChrY) and chromosome 21 (Chr21), which are associated with Alzheimer's disease (AD) pathology. This study investigates the role of DNA replication within centromeric regions of these chromosomes using human neural progenitor cells engineered to overexpress either wild-type (wt) or pseudo-hyper-phosphorylated (php) Tau protein. We developed a method to analyze replication dynamics in centromeric DNA. Our findings reveal that replication origins and fork pausing events are mainly located within α-satellite sequences of ChrY and Chr21, where wt and php Tau distinctly modulate origin activation and initiation. Mass spectrometry analysis on immunoprecipitated Tau identified nuclear interactors of Tau, particularly in its php form, which might directly influence the chromatin architecture and gene expression. These studies provide critical insights into the molecular mechanisms of aneuploidy in tauopathies.

Figure S4. Leading edge genes from GSEA showing downregulation of invasive phenotypes in TKO and DKOAA relative to DKO.

Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths. Mutations in the tumor-suppressor APC initiate CRC in part by preventing the glycogen synthase kinase 3 (GSK3) kinase from phosphorylating β-CATENIN, leading to its stabilization and transactivation of mitogenic target genes. While the importance of β-CATENIN phosphorylation by GSK3 is well established, APC regulation of GSK3 activity upon other targets is not understood. Here, we identify the H4K20 methyltransferase SETD8 as a target of APC-coordinated GSK3 phosphorylation in the intestinal epithelium. We find that phosphorylation by GSK3 restrains the oncogenic activity of SETD8, with loss of phosphorylation sensitizing mice to oncogenic insults. Mechanistically, loss of SETD8 phosphorylation in tumors results in a loss of H4K20 monomethylation (H4K20me1) deposition at oncogenic cholesterol biosynthesis and fetal intestinal genes, allowing for their activation in part through gain of YAP accessibility. These results underscore the importance of SETD8 in CRC and represent a novel β-CATENIN-independent oncogenic consequence of APC loss.