Abstract Chromosome segregation fidelity relies on stable attachments between kinetochores (KTs) and spindle microtubules during mitosis. We recently identified a previously unrecognized form of KT dysfunction that is prevalent in human glioblastoma (GBM) isolates and is triggered by aberrant activation of mitogen-activated protein kinase (MAPK) signaling during mitosis. This phenotype—termed MAPK-stressed kinetochores (MaSKs)—arises when hyperactive Ras-Raf-MEK-ERK signaling drives excessive phosphorylation of KT components by a network of KT-associated kinases. As a result, MT-binding affinity is reduced and KT-MT turnover becomes abnormally high, producing a lethal mitotic stress state. MaSKs provide a direct mechanistic link between oncogenic Ras/MAPK pathway activity and the generation of chromosome instability, filling a key gap in our understanding of how mitogenic oncogenes disrupt mitosis. Notably, MaSKs appear to be restricted to cancer and transformed cells, where they create unique genetic and molecular dependencies. MaSK-positive cells rely specifically on two non-essential domains of the mitotic checkpoint protein BubR1/BUB1B to recruit PP2A phosphatase and suppress MaSK-induced KT-MT instability. These dependencies suggest new opportunities for tumor-selective therapeutic targeting. In this presentation, we will describe the molecular basis of MaSK formation, the assays enabling their discovery, and evidence supporting their utility as biomarkers for a subset of MAPK-driven tumors. We further discuss how MaSKs can be exploited to identify novel therapeutic strategies and define a patient responder population. Citation Format: Jennifer DeLuca, Patrick J. Paddison. MAPK driven kinetochore instability as a biomarker and therapeutic vulnerability in glioblastoma [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 2979.

Glioblastoma (GBM) quiescent (Q) cell populations are hypothesized to contain cancer stem-like cells (CSC) that drive tumor growth, cellular heterogeneity, and recurrence. However, GBM tumors do not neatly resolve into developmental hierarchies and Q stem-like activities are difficult to assess. Here, we evaluated tumor Q subpopulations in patient-derived GBM xenograft tumors using live cell reporters, DNA label retention assays, and single cell genomics. Compared to adult neural stems cells (NSCs), GBM Q populations contain hybrid transcriptional states composed of networks found in both dormant and activated adult NSCs, resulting in constitutive expression of key Q egress transcription factors and their targets (e.g., AP-1 and CCND1/2). As a result, even the longest Q-residing cells (~12 days) in xenograft tumors continuously cycle and fail to enter dormant Q states. We provide evidence and hypothesize that transient Q states in primary tumors arise as part of distinct proliferative compartments rather than deterministic developmental hierarchies driven by CSC activity. We further speculate that increases in basal translation rates drive Q instability in GBM tumors.

N6-methyladenosine (m6A) is an abundant modification of mRNA with important regulatory roles in normal and malignant hematopoiesis. We previously reported that in human erythroid leukemia (HEL) cells, m6A mRNA marking selectively regulates translation of essential erythropoiesis genes required for in vitro differentiation and human erythroid colony formation. Here, we further investigated the timing and nature of requirement for m6A-methyltransferase (MTase) activity during human erythropoiesis, using a standardized in vitro erythroid differentiation assay for hHSPCs. We identified two critical m6A regulated developmental windows in BFU-E and during the transition from CFU-E to proerythroblasts. These windows of m6A-MTase requirement coincide with rising global m6A levels, which peak in proerythroblasts. After proerythroblast formation, however, m6A -MTase activity is dispensable for differentiation, proliferation, and survival. In BFU-E, m6A-MTase promotes proliferation but is dispensable for differentiation, while, in CFU-E, both m6A -MTase and the YTHDF family of m6A readers are essential for differentiation to proerythroblasts. Mechanistically, in CFU-E, m6A - MTase activity enhances translation of ribosomal and oxidative phosphorylation (OXPHOS) genes, thereby elevating global protein synthesis rates and enabling efficient erythroblast formation. We propose that this form of translational regulation by m6A emerged as an evolutionary adaptation to meet the high translational demands of human erythropoiesis.

Glioblastoma stem cell (GSC) cultures are initiated from glioblastoma (GBM) surgical resection tissue. When grown appropriately they can capture and propagate key GBM molecular and cellular features. We have characterized cellular, genomic and proteomic features of four isocitrate dehydrogenase (IDH)-expressing (IDH +) GSC cultures as cellular models for ~ 90% of adult GBMs. We demonstrate that GSC cultures can be continuously propagated in defined, serum-free media and 5% oxygen without specialized growth substrates; have culture-specific genomic and mtDNA variants together with gene/protein expression profiles; and display reproducible dose-survival curves for the GBM standard-of-care therapies ionizing radiation (IR) and temozolomide (TMZ). In order to better define GSC culture cellular heterogeneity and dynamics, we used lentiviral DNA barcoding, mtDNA variants and single cell gene expression profiling over 40 days after IR treatment. GSC cultures are versatile in their ability to support many in vitro protocols including high throughput screens as well as xenograft, organoid and other disease modeling protocols. They provide a simple cellular disease model for better understanding GBM biology, and for identifying new, potentially more effective GBM therapies and treatment regimens.

Single-cell transcriptomics has unveiled a vast landscape of cellular heterogeneity in which the cell cycle is a significant component. We trained a high-resolution cell cycle classifier (ccAFv2) using single cell RNA-seq (scRNA-seq) characterized human neural stem cells. The ccAFv2 classifies six cell cycle states (G1, Late G1, S, S/G2, G2/M, and M/Early G1) and a quiescent-like G0 state (Neural G0), and it incorporates a tunable parameter to filter out less certain classifications. The ccAFv2 classifier performed better than or equivalent to other state-of-the-art methods even while classifying more cell cycle states, including G0. We demonstrate that the ccAFv2 classifier effectively generalizes the S, S/G2, G2/M, and M/Early G1 states across cell types derived from all three germ layers. While the G0, G1, and Late G1 states perform well in neuroepithelial cell types, their accuracy is lower in other cell types. However, misclassifications are confined to the G0, G1, and Late G1 states. We showcased the versatility of ccAFv2 by successfully applying it to classify cells, nuclei, and spatial transcriptomics data in humans and mice, using various normalization methods and gene identifiers. We provide methods to regress the cell cycle expression patterns out of single cell or nuclei data to uncover underlying biological signals. The classifier can be used either as an R package integrated with Seurat or a PyPI package integrated with SCANPY. We proved that ccAFv2 has enhanced accuracy, flexibility, and adaptability across various experimental conditions, establishing ccAFv2 as a powerful tool for dissecting complex biological systems, unraveling cellular heterogeneity, and deciphering the molecular mechanisms by which proliferation and quiescence affect cellular processes.

Quiescence cancer stem-like cells may play key roles in promoting tumor cell heterogeneity and recurrence for many tumors, including glioblastoma (GBM). Here we show that the protein acetyltransferase KAT5 is a key regulator of transcriptional, epigenetic, and proliferative heterogeneity impacting transitions into G0-like states in GBM. KAT5 activity suppresses the emergence of quiescent subpopulations with neurodevelopmental progenitor characteristics, while promoting GBM stem-like cell (GSC) self-renewal through coordinately regulating E2F- and MYC- transcriptional networks with protein translation. KAT5 inactivation significantly decreases tumor progression and invasive behavior while increasing survival after standard of care. Further, increasing MYC expression in human neural stem cells stimulates KAT5 activity and protein translation, as well as confers sensitivity to homoharringtonine, to similar levels to those found in GSCs and high-grade gliomas. These results suggest that the dynamic behavior of KAT5 plays key roles in G0 ingress/egress, adoption of quasi-neurodevelopmental states, and aggressive tumor growth in gliomas.

Triple-negative breast cancer (TNBC, lacking expression of estrogen and progesterone receptors and amplification of HER2) is an aggressive and drug-resistant subtype. Current standard-of-care for early-stage disease with neoadjuvant chemoimmunotherapy results in a pathologic complete response in only ∼60% of patients. Proliferative heterogeneity naturally occurs in most tumors, where tumor cells exist in multiple different states with varying proliferative potential—states of active division, long- and short-term quiescence, pre-cell cycle entry, and stress. TNBC tumors exhibit a higher percentage of actively cycling, or Ki-67–positive cells, compared to other molecular subsets of breast cancer, which has been shown to predict higher pathologic complete response rates following neoadjuvant chemotherapy. Unfortunately, despite this, TNBC patients experience worse overall survival rates driven by higher rates of relapse. It has been proposed that a significant portion of noncycling, or Ki-67–negative, cells are in a G0-like, or quiescent, state and predicted therefore to be resistant to cytotoxic chemotherapies that rely on actively dividing cells. The presence of a tumor cell population in a quiescent/G0-like state has been increasingly recognized across many tumor types, including in breast cancer. Patient-derived tumor organoids have emerged as an appealing model system that retains more cellular heterogeneity from primary tumors compared to widely used two-dimensional cell lines. Organoids show remarkable complexity by single-cell RNA sequencing, representing many more cell states compared to best available two-dimensional cell lines that most closely resemble the mutation, copy number variation, gene expression and protein expression profiles of patient tumors. A mutant version of p27 containing mutations blocking binding to cyclin/cdk complexes, but retained proteolysis in S/G2/M, combined with an mVenus fluorescent protein (p27-mVenus) has been previously validated as a genetic reporter for steady-state G0 readouts. We have optimized lentiviral transduction of a triple-negative breast cancer patient-derived xenograft organoid with the p27-mVenus reporter to label quiescent organoid subpopulations. Optimized parameters include method of lentivirus concentration, multiplicity of infection, duration of transduction, presence of protamine, presence of a Matrigel base layer, presence of RetroNectin and use of spinoculation. We will use this p27-mVenus TNBC organoid reporter line to study the response of quiescent cell populations to current standard of care treatments and how they contribute to therapeutic resistance. Citation Format: Darien Reed-Perino, Sonali Arora, Alana L Welm, Cyrus M Ghajar, Patrick J Paddison. Optimization of lentiviral transduction of a triple-negative breast cancer patient-derived xenograft organoid for modeling tumor cell quiescence and associated treatment resistance [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-04-21.

Abstract Gliomas are the most common type of brain tumor in both children and adults. Communication between glioma cells and the brain tumor microenvironment (TME) is a fundamental aspect of brain cancer pathophysiology. Glioma cells secrete factors that act as chemoattractants, influencing the TME. In addition, neuronal activity within the TME drives the proliferation and growth of gliomas through paracrine signaling, with brain-derived neurotrophic factor (BDNF) being a key mediator. BDNF binds with high affinity to the tropomyosin receptor kinase B (TrkB). Recent work from our group has demonstrated that the TrkB.T1 splice variant is upregulated in human gliomas. Furthermore, TrkB.T1 overexpression enhances tumor aggressiveness in vivo and is associated with the downregulation of genes involved in tumor cell recognition and elimination. However, the role of the TrkB.T1 splice variant in the bidirectional communication between glioma cells and the surrounding microenvironment remains unexplored. Our aim was to investigate whether the increased levels of TrkB.T1 observed in gliomas contribute to modulating immune responses. We investigated the immune cell heterogeneity associated with elevated TrkB.T1 levels in vivo using a glioma mouse model engineered with RCAS/tv-a technology. Tumors overexpressing TrkB.T1 exhibited increased neutrophil recruitment, a phenomenon linked to tumor growth, metastasis and therapeutic resistance. To further dissect the signaling mechanisms underlying the BDNF-TrkB.T1 axis, we conducted in vitro studies using glioma stem cells (GSCs) that express high levels of TrkB.T1. GSCs were treated with BDNF, after which the conditioned media was analyzed using a cytokine multiplex assay, and cell lysates were subjected to proteomic analysis. Our findings revealed that TrkB.T1 significantly upregulates key chemokines previously implicated in neutrophil recruitment. Moreover, BDNF signaling increased the expression of N-cadherin, β- catenin, and Snail, markers indicative of the epithelial-to-mesenchymal transition (EMT) phenotype. To further explore the role of TrkB.T1, we used CRISPR/Cas9-based genome editing to knock down TrkB.T1 in GSCs. In conclusion, our findings suggest that the highly expressed TrkB.T1 receptor in gliomas contributes to creating an immunosuppressive microenvironment by recruiting neutrophils and promoting EMT. These insights highlight TrkB.T1 as a potential therapeutic target for modulating glioma-immune cell interactions, which could be crucial in curbing disease progression. Citation Format: Leyre Merino-Galan, Sergio Ortiz-Espinosa, Hawa L. Jagana, John M. Hemenway, Ashmitha Rajendran, Taylor S. Jackson, Daniel A. Kuppers, Sonali Arora, Patrick J. Paddison, Siobhan S. Pattwell. BDNF-TrkB.T1 signaling increases neutrophil recruitment and epithelial-to- mesenchymal transition in gliomas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B043.

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. To identify genes differentially required for the viability of GBM stem-like cells (GSCs), we performed functional genomic lethality screens comparing GSCs and control human neural stem cells. Among top scoring hits in a subset of GBM cells was the F-box-containing gene FBXO42 , which was also essential in ∼15% of cell lines derived from a broad range of cancers. Mechanistic studies revealed that, in sensitive cells, FBXO42 activity prevents chromosome alignment defects, mitotic cell cycle arrest, and cell death. The cell cycle arrest, but not the cell death, triggered by FBXO42 inactivation could be suppressed by brief exposure to a chemical inhibitor of Mps1, a key spindle assembly checkpoint (SAC) kinase. FBXO42 ’s cancer-essential function requires its F-box and Kelch domains, which are necessary for FBXO42’s substrate recognition and targeting by SCF ubiquitin ligase complex. However, none of FBXO42’s previously proposed targets, including ING4, p53, and RBPJ, were responsible for the observed phenotypes. Instead, our results suggest that FBOX42 activity suppresses the accumulation of one or more proteins that perturb chromosome-microtubule dynamics in cancer cells, which, in turn, leads to induction of the SAC and cell death.

Glioblastoma is universally fatal and characterized by frequent chromosomal copy number alterations harboring oncogenes and tumor suppressors. In this study, we analyzed exome-wide human glioblastoma copy number data and found that cytoband 6q27 is an independent poor prognostic marker in multiple data sets. We then combined CRISPR-Cas9 data, human spatial transcriptomic data, and human and mouse RNA sequencing data to nominate PDE10A as a potential haploinsufficient tumor suppressor in the 6q27 region. Mouse glioblastoma modeling using the RCAS/tv-a system confirmed that Pde10a suppression induced an aggressive glioma phenotype in vivo and resistance to temozolomide and radiation therapy in vitro. Cell culture analysis showed that decreased Pde10a expression led to increased PI3K/AKT signaling in a Pten-independent manner, a response blocked by selective PI3K inhibitors. Single-nucleus RNA sequencing from our mouse gliomas in vivo, in combination with cell culture validation, further showed that Pde10a suppression was associated with a proneural-to-mesenchymal transition that exhibited increased cell adhesion and decreased cell migration. Our results indicate that glioblastoma patients harboring PDE10A loss have worse outcomes and potentially increased sensitivity to PI3K inhibition.

PDF file - 340K, Effects of BUB1B knockdown on in human astrocytes, RasV12 transformed astrocytes, Hela cells and RPE-1 cells

PDF file - 133K, BTIC and NSC growth phenotypes resulting from knockdown of PLK1 and pharmacological inhibition of Aurora B kinase

Abstract Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). In this study, we perform an epigenetic-focused functional genomics screen in GBM organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche. Despite their importance for tumor growth, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy resistant niche. Our niche-specific screens identified WDR5, an H3K4 histone methyltransferase responsible for activating specific gene expression, as indispensable for GBM CSC growth and survival. In GBM CSC models, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, required for stem cell maintenance and including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment resulted in dose-dependent silencing of stem cell reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSC in vitro growth and self-renewal as well as in vivo tumor growth. Our results unveiled the role of WDR5 in maintaining the CSC state in GBM and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers. This conceptual and experimental framework can be applied to many cancers, and can unmask unique microenvironmental biology and rationally designed combination therapies.

It is clear from the high mortality rate in advanced cancers that we need a more complete understanding of the many sources of oncogenic dysregulation in tumors. The importance of the non-coding genome in disease has recently begun to be revealed through expanded use of whole genome sequencing. In particular, 3’ untranslated region (3’UTR) somatic mutations represent an important but largely unexplored avenue of alternative oncogenic gene dysregulation. Individual instances of 3’UTR-mediated oncogenicity are known, including oncogenic RNA binding proteins and microRNAs. However, a comprehensive, high-throughput study of patient-based 3’UTR mutations and their effect on post-transcriptional gene regulation in cancer has yet to be undertaken. To determine the significance of 3’UTR mutations in advanced disease, we identify 3’UTR somatic variants across 185 metastatic castration-resistant prostate tumors, discovering 14,497 single-nucleotide mutations, which are enriched in oncogenic pathways and 3’UTR regulatory elements. We develop two complementary massively parallel reporter assays (MPRAs) to measure how thousands of these patient-based mutations affect two distinct levels of post-transcriptional gene regulation: mRNA translation and stability. These MPRAs identify hundreds of functional variants that allow us to define three determinants of mutation significance: sequence conservation, known regulatory elements, and RNA structure. Furthermore, we demonstrate the clinical relevance of these mutations, observing that CRISPR-Cas9 base editing of distinct patient 3’UTR mutations into endogenous cellular loci increases oncogenic mRNA translation and cellular stress resistance. Finally, we go back to patients, illustrating that those harboring oncogenic 3’UTR mutations discovered by our methods display particularly poor prognosis. This work represents an unprecedented view of the extent to which disease-relevant 3’UTR mutations affect mRNA stability, translation, and cancer progression, uncovering principles of regulatory functionality and potential therapeutic targets in previously unexplored regulatory regions. Citation Format: Samantha L. Schuster, Sonali Arora, Cynthia L. Wladyka, Lukas Corey, Bethany L. Stackhouse, Lori Kollath, Eva Corey, Lawrence D. True, Dave Young, Patrick J. Paddison, Andrew C. Hsieh. Multi-level functional genomics reveals molecular and cellular oncogenicity of patient-based 3’ untranslated region mutations [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR005.

Therapy for glioblastoma (GBM) includes surgical resection, radiation, and chemotherapy, which functionally reduces tumor burden to a state of minimal residual disease (MRD). However, recurrence over time is universal. Large-scale longitudinal studies of GBM patient samples did not identify selection pressure for specific DNA mutations in recurrent disease, highlighting the importance of epigenetic mechanisms during relapse. In this study, we aim to model MRD and the functional and transcriptional effects of epigenetic perturbations in GBM patient derived xenografts (PDXs). We developed an in vivo inducible genome editing system by engineering 827 GBM stem cells to express doxycycline inducible Cas9 and a pool of 14 CRISPR sgRNAs targeting 5 epigenetic factors (readers/erasers of H3K27me3 and H4 acetyltransferase KAT5) and controls. Tumor cells are implanted orthotopically and mice are followed by serial MRIs until tumor volumes reach 2mm3, after which in vivo editing is induced by administration of doxycycline. Cells are harvested for scRNA-sequencing and targeted amplification of sgRNA libraries, allowing for assignment of sgRNAs to individual cells. Genome editing efficiencies ranged from 50-90% in vivo. Knockout of core Polycomb (PcG) complex members led to dysregulated expression of genes involved in therapeutic resistance and altered cell state distributions. Current directions include optimization of sgRNA targeting and recovery of CRISPR-edited cells from tumors for scaling up screens. In addition, we have developed a “standard of care” treatment regimen in GBM PDX models whereby temozolomide and radiation therapy is dose optimized to achieve a latent state that mimics MRD followed by eventual tumor recurrence. Future experiments will combine MRD modeling in PDXs with in vivo CRISPR screening to characterize functional dependencies of MRD in GBM. This data provides proof of concept for utilizing these tools for studying genotype to phenotype connections in a complex GBM model system.

It is now well established from single-cell RNA-seq studies that glioblastoma (GBM) tumors are complex, maligned neuro-developmental ecosystems harboring diverse tumor cell types. Neoplastic cells can resemble astrocytes, neural progenitors, oligodendrocyte progenitor cells, mesenchymal cells, and radial glial cells that contribute to tumor growth and homeostasis in specific ways. However, GBM single-cell data sets have failed to produce general models for transitions in and out of specific developmental and proliferative states in tumors. One reason is that human GBM tumors do not neatly resolve into developmental hierarchies. Here we focused on modeling GBM tumor cellular heterogeneity by defining "proliferative compartments" in single-cell transcriptomic data derived from primary tumors and early passage tumorsphere cultures. Previously, we observed that each tumor cell entering S-phase has a unique developmental signature that can be leveraged to define broader partitioned proliferative compartments (PPCs) with distinct developmental gene expression and genomic alteration patterns. Thus, we extracted the S-phase cells from a tumor, defined the proliferative compartments in that tumor using de novo clustering, determined the marker genes for each compartment, and defined the broader PPCs across tumors by de novo clustering based on similarity in marker genes. From a cohort of six tumors we observed eight broader PPCs and found that tumors can contain as many as five PPCs or as few as two. Because tumor growth and recurrence both require cell proliferation, we propose that patient-specific PPCs represent the engines of GBM progression.

PDF file - 1MB, Additional information showing cancer cells have added requirement on BUB1B for chromosome alignment and KT-MT attachment

PDF file - 134K, Ras-Mapk signaling is inappropriately high BTICs and NHA-Ras cells

Abstract In solid tumors, G0-like states are likely critical for maintaining developmental hierarchies and cellular heterogeneity and promoting tumor growth/recurrence, yet little is known about tumor G0 states or regulation of their ingress/egress. To discover G0-like states and their regulators for glioblastoma (GBM), we previously performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) for genes that trap cells in G0 when inhibited. We identify the protein acetyltransferase KAT5 as a key regulator of GBM G0 ingress/egress. Here, we show in an in vivo GSC-derived orthotopic xenograft model that KAT5 regulates transcriptional, epigenetic, and proliferative heterogeneity impacting transitions into G0-like states. We show that KAT5 activity suppresses the emergence of non-dividing subpopulations with oligodendrocyte progenitor and radial glial cell characteristics. With regard to chromatin regulation, we show that loss of KAT5 activity alters sites of mixed epigenetic valency (which have both activating and repressive marks in bulk tumor populations) to promote emergence of OPC/RG G0-like populations in tumors. Our data reveal KAT5-associated transcriptional and epigenetic changes in tumors, which indicate that KAT5 activity biases cell state distribution away from OPC and RG-like subpopulations. In addition, loss of KAT5 resulted in a less invasive phenotype and prolonged survival of mice harboring orthotopic xenografts.