Biology subjects

Iacobuzio-Donahue, C. A.

Publications and source records attributed to Iacobuzio-Donahue, C. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Hierarchical classification of immune cell transcriptomes at population-scale

Accurate immune cell classification is essential for interpreting single-cell RNA sequencing (scRNA-seq) data. However, progress in automating cell type annotation is constrained by the lack of independent, high-resolution benchmarks, as routine data integration introduces statistical dependencies that inflate model generalizability. Here, we present the single-cell universal classification omnibus (Suco), a resource of independent, uniform expert annotations, and Compocyte, a modular hierarchical classifier. Together, they establish a framework that substantially outperforms existing classifiers while facilitating expert review of ambiguous annotations. Applying Compocyte across 50 studies, including three newly generated datasets, we classified 15.6 million leukocytes from 3,965 patients. Within this cohort, we identified a new tumor-associated resorptive macrophage phenotype, a non-canonical monocyte subtype in subclinical cytokine release syndrome, and the programmatic erosion of T cell memory stemness across metastatic sites. Suco and Compocyte thus provide a generalizable framework to uncover the principles governing human immunity at population scale. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/728980v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@13a6e4borg.highwire.dtl.DTLVardef@11f1626org.highwire.dtl.DTLVardef@1e72bd4org.highwire.dtl.DTLVardef@1ee799b_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefThe single-cell universal classification omnibus and the modular hierarchical classifier Compocyte enable annotating single cell RNA sequencing data from 3,965 patients, revealing novel resorptive macrophage and vaccination-associated monocyte states, alongside the erosion of T cell memory stemness as a hallmark of solid tumor metastases. HighlightsO_LISuco, a benchmark enabling novel single cell artificial intelligence models C_LIO_LICompocyte, a hierarchical cell type classifier outperforming current architectures C_LIO_LIMacrophages adopt osteoclast-like gene expression states across cancer types C_LIO_LIStem-like programs erode in metastasis-infiltrating T memory cells across tumors C_LI

bioinformatics↗

bioRxiv2026

POLQ-driven repair scars shape the immunogenic landscape of homologous recombination-deficient pancreatic cancer

Pancreatic cancer (PC) is broadly resistant to immune checkpoint blockade, although a subset of homologous recombination-deficient (HRD) tumors exhibits durable immune engagement. The genomic features that distinguish these immune-responsive tumors from immune-inert HRD tumors remain poorly understood. Here we identify a microhomology-mediated end joining (MMEJ) repair scar, the MMEJ Deletion Footprint (MDF), as a genomic readout of POLQ-associated error-prone repair that enriches for frameshift indels. Across the multi-omic discovery cohort integrating tumor genomics, single-nucleus transcriptomics and spatial immune profiling, MDF-high HRD PC exhibited increased frameshift-indel-derived neoantigens and interferon programs. MDF was further associated with remodeling of the myeloid compartment toward MHC II-high dendritic cell-like antigen-presenting macrophage states and the immune synapse architecture marked by increased spatial interaction between APC-like macrophages and cytotoxic CD8+ T cells. These tissue-level features aligned with a functional trajectory shift of CD8+ T cells, consistent with effective anti-tumor immunity and was associated with favorable clinical outcomes of patients. Together, our findings position MMEJ-linked repair scarring as actionable biology that connects an HRD genotype to immune organization and suggests rational immunotherapy combinations that may enhance antigen presentation and myeloid activation to extend durable benefit in HRD-lineage cancers.

cancer biology↗

bioRxiv2026

A Genetically Engineered Human Organoid Model Reveals Distinct Genetic and Epigenetic Barriers of Lineage Plasticity in Early PDAC Transformation

The lack of accurate, human-based models recapitulating early-stage pancreatic ductal adenocarcinoma (PDAC) has hindered therapeutic development. Using pluripotent stem cell-derived pancreatic progenitor organoids, we established a human PDAC model that faithfully reproduces the genetic, epigenetic, and transcriptomic trajectory of tumor initiation and progression in vitro, validated against clinical datasets and histopathology. We demonstrate that CDKN2A loss, nearly universal in patients but dispensable in mouse models, is essential for neoplastic transformation when combined with KRAS and TP53 mutations, while SMAD4 loss promotes tumor progression. Multi-omics profiling reveals epigenetic repression of pancreatic lineage program during PDAC initiation, alongside oncogenic AP-1-driven chromatin remodeling. Notably, we identify TET1 suppression as a mechanistic link between oncogenic ERK signaling and the hypermethylation and silencing of essential pancreatic transcription factors. This model captures the genetic and epigenetic determinants of human PDAC, reveals antagonism between oncogenic and lineage restriction programs, and supports TET-based lineage restoration as a promising early intervention strategy for high-risk individuals.

cancer biology↗

bioRxiv2025

SPINK1-COL18A1 Crosstalk Shapes Epigenome and Drives Cancer Stemness in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer with an increasing incidence and extremely dismal prognosis. Discovery and mechanistic understanding of the genetic and epigenetic drivers of PDAC is therefore of critical importance. Here, we uncover serine protease inhibitor Kazal type 1 (SPINK1) as a putative determinant of clinical progression and demonstrate that it can have a non-canonical function as a regulator of epigenomic states of PDAC cells and associated extensive changes in gene expression. We show that SPINK1 expression, which varies in PDAC, is associated with key aggressive phenotypic cancer states and is correlated with the expression of stemness markers both in vitro and in patient samples. Mechanistically, our results strongly suggest that SPINK1 acts through a new signaling axis, by interacting with COL18A1 in the Golgi apparatus, promoting endostatin release and eventually inducing extensive histone H3 modifications. These results reveal a new function of SPINK1 in PDAC and suggest a potential therapeutic route to combat PDAC aggressiveness by targeting the SPINK1-COL18A1-endostatin signaling.

cancer biology↗

bioRxiv2025

Transcriptomic plasticity is a hallmark of metastatic pancreatic cancer

Metastasis is the leading cause of cancer deaths; nonetheless, how tumor cells adapt to vastly different organ contexts is largely unknown. To investigate this question, we generated a transcriptomic atlas of primary tumor and diverse metastatic samples from a patient with pancreatic ductal adenocarcinoma who underwent rapid autopsy. Unsupervised archetype analysis identified both shared and site-specific gene programs, including lipid metabolism and gastrointestinal programs prevalent in peritoneum and stomach wall lesions, respectively. We developed a probabilistic approach for inferring clonal phylogeny from single-cell and matched whole-exome data. Distantly related genetic clones in the peritoneum express the lipid metabolism program, likely due to signaling by the adipocyte-rich peritoneum environment, and cells in most clones express multiple programs, suggesting that transcriptomic plasticity is a prevalent feature of metastatic cells. These deeply annotated analyses using a patient-centric platform provide a model for investigating metastatic mechanisms and plasticity in advanced cancer.

bioinformatics↗

bioRxiv2024

Genomic evolution of pancreatic cancer at single-cell resolution

We adapted a previously developed targeted single-nucleus DNA sequencing (snDNA-seq) method and constructed a new suite of computational analysis tools to study 137,491 single-nucleus DNA libraries from 24 pancreatic cancers collected under a variety of clinical scenarios including early and late diagnoses, different metastatic sites and before- and after-treatment. We refined the mutational landscape of pancreatic cancer by capturing events missed by bulk sequencing, and validated the evolution pattern of early fixation of driver single-nucleotide variants (SNVs) followed by generation of intratumoral heterogeneity for copy number variations (CNVs). Intertumoral convergent evolution was common, including subclonal inactivation of TGF-{beta} pathway by mutating various components of it; intratumoral convergence was rarely observed, likely due to strong selective force in pancreatic cancer development. Continuous evolution was frequently seen manifesting as CNVs. In the context of non-targeted treatments, no particular pattern was found across metastases or through treatment. In six pancreatic cancers with germline BRCA2 mutation, we discovered varied timing of biallelic inactivation of BRCA2, which sculpted different evolutionary trajectories and could presumably contribute to differential response to treatment. As the first large-scale application of targeted snDNA-seq on pancreatic cancer, this work provides a sample processing and computational analysis pipeline that warrants further clinical utility.

cancer biology↗

bioRxiv2024

Mechanism of neurodegeneration mediated by clonal inflammatory microglia

Langerhans cell Histiocytosis (LCH) and Erdheim-Chester disease (ECD) are clonal myeloid disorders, associated with MAP-Kinase activating mutations and an increased risk of neurodegeneration. Surprisingly, we found pervasive PU.1+ microglia mutant clones across the brain of LCH and ECD patients with and without neurological symptoms, associated with microgliosis, reactive astrocytosis, and neuronal loss. The disease predominated in the grey nuclei of the rhombencephalon, a topography attributable to a local proliferative advantage of mutant microglia. Presence of clinical symptoms was associated with a longer evolution of the disease and a larger size of PU.1+ clones (p= 0.0003). Genetic lineage tracing of PU.1+ clones suggest a resident macrophage lineage or a bone marrow precursor origin depending on patients. Finally, a CSF1R-inhibitor depleted mutant microglia and limited neuronal loss in mice suggesting an alternative to MAPK inhibitors. These studies characterize a progressive neurodegenerative disease, caused by clonal proliferation of inflammatory microglia (CPIM), with a decade(s)-long preclinical stage of incipient disease that represent a therapeutic window for prevention of neuronal death.

immunology↗

bioRxiv2024

Metastatic site influences driver gene function in pancreatic cancer

Driver gene mutations can increase the metastatic potential of the primary tumor1-3, but their role in sustaining tumor growth at metastatic sites is poorly understood. A paradigm of such mutations is inactivation of SMAD4 - a transcriptional effector of TGF{beta} signaling - which is a hallmark of multiple gastrointestinal malignancies4,5. SMAD4 inactivation mediates TGF{beta}s remarkable anti-to pro-tumorigenic switch during cancer progression and can thus influence both tumor initiation and metastasis6-14. To determine whether metastatic tumors remain dependent on SMAD4 inactivation, we developed a mouse model of pancreatic ductal adenocarcinoma (PDAC) that enables Smad4 depletion in the pre-malignant pancreas and subsequent Smad4 reactivation in established metastases. As expected, Smad4 inactivation facilitated the formation of primary tumors that eventually colonized the liver and lungs. By contrast, Smad4 reactivation in metastatic disease had strikingly opposite effects depending on the tumors organ of residence: suppression of liver metastases and promotion of lung metastases. Integrative multiomic analysis revealed organ-specific differences in the tumor cells epigenomic state, whereby the liver and lungs harbored chromatin programs respectively dominated by the KLF and RUNX developmental transcription factors, with Klf4 depletion being sufficient to reverse Smad4s tumor-suppressive activity in liver metastases. Our results show how epigenetic states favored by the organ of residence can influence the function of driver genes in metastatic tumors. This organ-specific gene-chromatin interplay invites consideration of anatomical site in the interpretation of tumor genetics, with implications for the therapeutic targeting of metastatic disease.

cancer biology↗

bioRxiv2024

Islands of genomic stability in the face of genetically unstable metastatic cancer

AbstractO_ST_ABSIntroductionC_ST_ABSMetastatic cancer affects millions of people worldwide annually and is the leading cause of cancer-related deaths. Most patients with metastatic disease are not eligible for surgical resection, and current therapeutic regimens have varying success rates, some with 5-year survival rates below 5%. Here we test the hypothesis that metastatic cancer can be genetically targeted by exploiting single base substitution mutations unique to individual cells that occur as part of normal aging prior to transformation. These mutations are targetable because [~]10% of them form novel tumor-specific "NGG" protospacer adjacent motif (PAM) sites targetable by CRISPR-Cas9. MethodsWhole genome sequencing was performed on five rapid autopsy cases of patient-matched primary tumor, normal and metastatic tissue from pancreatic ductal adenocarcinoma decedents. CRISPR-Cas9 PAM targets were determined by bioinformatic tumor-normal subtraction for each patient and verified in metastatic samples by high-depth capture-based sequencing. ResultsWe found that 90% of PAM targets were maintained between primary carcinomas and metastases overall. We identified rules that predict PAM loss or retention, where PAMs located in heterozygous regions in the primary tumor can be lost in metastases (private LOH), but PAMs occurring in regions of loss of heterozygosity (LOH) in the primary tumor were universally conserved in metastases. ConclusionsRegions of truncal LOH are strongly retained in the presence of genetic instability, and therefore represent genetic vulnerabilities in pancreatic adenocarcinomas. A CRISPR-based gene therapy approach targeting these regions may be a novel way to genetically target metastatic cancer.

cancer biology↗

bioRxiv2024

A microglia clonal inflammatory disorder in Alzheimer's Disease

Somatic genetic heterogeneity resulting from post-zygotic DNA mutations is widespread in human tissues and can cause diseases, however few studies have investigated its role in neurodegenerative processes such as Alzheimers Disease (AD). Here we report the selective enrichment of microglia clones carrying pathogenic variants, that are not present in neuronal, glia/stromal cells, or blood, from patients with AD in comparison to age-matched controls. Notably, microglia-specific AD-associated variants preferentially target the MAPK pathway, including recurrent CBL ring-domain mutations. These variants activate ERK and drive a microglia transcriptional program characterized by a strong neuro-inflammatory response, both in vitro and in patients. Although the natural history of AD-associated microglial clones is difficult to establish in human, microglial expression of a MAPK pathway activating variant was previously shown to cause neurodegeneration in mice, suggesting that AD-associated neuroinflammatory microglial clones may contribute to the neurodegenerative process in patients. One-Sentence SummaryA subset of Alzheimer Disease patients carry mutant microglia somatic clones which promote neuro-inflammation.

neuroscience↗

bioRxiv2023

Cancer cells co-evolve with retrotransposons to mitigate viral mimicry

Overexpression of repetitive elements is an emerging hallmark of human cancers1. Diverse repeats can mimic viruses by replicating within the cancer genome through retrotransposition, or presenting pathogen-associated molecular patterns (PAMPs) to the pattern recognition receptors (PRRs) of the innate immune system2-5. Yet, how specific repeats affect tumor evolution and shape the tumor immune microenvironment (TME) in a pro- or anti-tumorigenic manner remains poorly defined. Here, we integrate whole genome and total transcriptome data from a unique autopsy cohort of multiregional samples collected in pancreatic ductal adenocarcinoma (PDAC) patients, into a comprehensive evolutionary analysis. We find that more recently evolved Short Interspersed Nuclear Elements (SINE), a family of retrotransposable repeats, are more likely to form immunostimulatory double-strand RNAs (dsRNAs). Consequently, younger SINEs are strongly co-regulated with RIG-I like receptor associated type-I interferon genes but anti-correlated with pro-tumorigenic macrophage infiltration. We discover that immunostimulatory SINE expression in tumors is regulated by either Long Interspersed Nuclear Elements 1 (LINE1/L1) mobility or ADAR1 activity in a TP53 mutation dependent manner. Moreover, L1 retrotransposition activity tracks with tumor evolution and is associated with TP53 mutation status. Altogether, our results suggest pancreatic tumors actively evolve to modulate immunogenic SINE stress and induce pro-tumorigenic inflammation. Our integrative, evolutionary analysis therefore illustrates, for the first time, how dark matter genomic repeats enable tumors to co-evolve with the TME by actively regulating viral mimicry to their selective advantage.

cancer biology↗

bioRxiv2023

ConDoR: Tumor phylogeny inference with a copy-number constrained mutation loss model

Tumors consist of subpopulations of cells that harbor distinct collections of somatic mutations. These mutations range in scale from single nucleotide variants (SNVs) to large-scale copy-number aberrations (CNAs). While many approaches infer tumor phylogenies using SNVs as phylogenetic markers, CNAs that overlap SNVs may lead to erroneous phylogenetic inference. Specifically, an SNV may be lost in a cell due to a deletion of the genomic segment containing the SNV. Unfortunately, no current single-cell DNA sequencing (scDNA-seq) technology produces accurate measurements of both SNVs and CNAs. For instance, recent targeted scDNA-seq technologies, such as Mission Bio Tapestri, measure SNVs with high fidelity in individual cells, but yield much less reliable measurements of CNAs. We introduce a new evolutionary model, the constrained k-Dollo model, that uses SNVs as phylogenetic markers and partial information about CNAs in the form of clustering of cells with similar copy-number profiles. This copy-number clustering constrains where loss of SNVs can occur in the phylogeny. We develop ConDoR (Constrained Dollo Reconstruction), an algorithm to infer tumor phylogenies from targeted scDNA-seq data using the constrained k-Dollo model. We show that ConDoR outperforms existing methods on simulated data. We use ConDoR to analyze a new multi-region targeted scDNA-seq dataset of 2153 cells from a pancreatic ductal adenocarcinoma (PDAC) tumor and produce a more plausible phylogeny compared to existing methods that conforms to histological results for the tumor from a previous study. We also analyze a metastatic colorectal cancer dataset, deriving a more parsimonious phylogeny than previously published analyses and with a simpler monoclonal origin of metastasis compared to the original study. Code availabilitySoftware is available at https://github.com/raphael-group/constrained-Dollo

bioinformatics↗

bioRxiv2023

Subclonal somatic copy number alterations emerge and dominate in recurrent osteosarcoma

Multiple large-scale tumor genomic profiling efforts have been undertaken in osteosarcoma, however, little is known about the spatial and temporal intratumor heterogeneity and how it may drive treatment resistance. We performed whole-genome sequencing of 37 tumor samples from eight patients with relapsed or refractory osteosarcoma. Each patient had at least one sample from a primary site and a metastatic or relapse site. We identified subclonal copy number alterations in all but one patient. We observed that in five patients, a subclonal copy number clone from the primary tumor emerged and dominated at subsequent relapses. MYC gain/amplification was enriched in the treatment-resistant clone in 6 out of 7 patients with more than one clone. Amplifications in other potential driver genes, such as CCNE1, RAD21, VEGFA, and IGF1R, were also observed in the resistant copy number clones. Our study sheds light on intratumor heterogeneity and the potential drivers of treatment resistance in osteosarcoma. SignificanceSubclonal copy number clones emerged and dominated in relapsed osteosarcoma, with MYC gain/amplification being the defining characteristic in our cohort. Selective pressure from neoadjuvant chemotherapy revealed this clone at the time of primary resection, highlighting that genomic profiling at this time may identify clones that are selected for, or determine innate resistance to primary chemotherapy.

cancer biology↗

bioRxiv2022

Chromosome 9p21.3 Coordinates Cell Intrinsic and Extrinsic Tumor Suppression

Somatic chromosomal deletions are prevalent in cancer, yet their functional contributions remain ill-defined. Among the most prominent of these events are deletions of chromosome 9p21.3, which disable a cell intrinsic barrier to tumorigenesis by eliminating the CDKN2A/B tumor suppressor genes. However, half of 9p21.3 deletions encompass a cluster of 16 type I interferons (IFNs) whose co-deletions have not been functionally characterized. To dissect how 9p21.3 and other genomic deletions impact cancer, we developed MACHETE (Molecular Alteration of Chromosomes with Engineered Tandem Elements), a genome engineering strategy that enables flexible modeling of megabase-sized deletions. Generation of 9p21.3-syntenic deletions in a mouse model of pancreatic cancer revealed that concomitant loss of Cdkn2a/b and the IFN cluster led to immune evasion and metastasis compared to Cdkn2a/b-only deletions. Mechanistically, IFN co-deletion disrupted type I IFN signaling, altered antigen-presenting cells, and facilitated escape from CD8+ T cell surveillance in a cell extrinsic manner requiring loss of interferon epsilon (Ifne). Our results establish co-deletions of the IFN cluster as a pervasive route to tumor immune evasion and metastasis, revealing how deletions can disable physically linked cell intrinsic and extrinsic tumor suppression. Our study establishes a framework to dissect the functions of genomic deletions in cancer and beyond.

cancer biology↗

bioRxiv2022

Application of high-throughput, high-depth, targeted single-nucleus DNA sequencing in pancreatic cancer

Despite insights gained by bulk DNA sequencing of cancer it remains challenging to resolve the admixture of normal and tumor cells, and/or of distinct tumor subclones; high throughput single-cell DNA sequencing circumvents these and brings cancer genomic studies to higher resolution. However, its application has been limited to liquid tumors or a small batch of solid tumors, mainly because of the lack of a scalable workflow to process solid tumor samples. Here we optimized a highly automated nuclei extraction workflow that achieved fast and reliable targeted single-nucleus DNA library preparation of 38 samples from 16 pancreatic adenocarcinoma (PDAC) patients, with an average library yield per sample of 2867 single nuclei. We demonstrate that this workflow not only performs well using low cellularity or low tumor purity samples but reveals novel genomic evolution patterns of PDAC as well.

cancer biology↗

bioRxiv2021

BRCA mutational status shapes the stromal microenvironment of pancreatic cancer linking CLU+ CAF expression with HSF1 signaling

Cancer-associated fibroblasts (CAFs) give rise to desmoplastic stroma, which supports tumor progression and metastasis, and comprises up to 90% of the tumor mass in pancreatic cancer. Recent work by us and others has shown that CAFs are transcriptionally rewired by adjacent cancer cells to form heterogeneous subtypes. Whether this rewiring is differentially affected by different driver mutations in cancer cells is largely unknown. Here we address this question by dissecting and comparing the stromal landscape of BRCA-mutated and BRCA Wild-type (WT) pancreatic ductal adenocarcinoma (PDAC). We comprehensively analyze PDAC samples from a cohort of 42 patients by laser-capture microdissection, RNA-sequencing and multiplexed immunofluorescence, revealing different CAF subtype compositions in germline BRCA-mutated vs. BRCA-WT tumors. In particular, we detect an increase in a subset of Clusterin (CLU)-positive CAFs in BRCA-mutated tumors. We further unravel a network of stress responses upregulated in BRCA-mutated tumors. Using cancer organoids and cell co-cultures, we show that the transcriptional shift of pancreatic stellate cells into CLU+ CAFs is mediated through activation of heat-shock factor 1 (HSF1), the transcriptional regulator of Clu. Our findings unravel a new dimension of stromal heterogeneity, influenced by germline mutations in cancer cells, with direct translational implications for clinical research. SignificanceBRCA1/2 mutations initiate some of the deadliest cancers, yet the fibroblastic microenvironment of BRCA-mutated cancers remains uncharted. Our work addresses a major unsolved question - to what extent is the tumor microenvironment determined by cancer mutations? We find that BRCA mutations in the cancer cells affect the composition of CAFs in PDAC. These findings have direct implications for diagnosis and for efforts to exploit CAFs for therapy.

cancer biology↗

bioRxiv2021

Genomic characterization of metastatic patterns from prospective clinical sequencing of 25,000 patients

Progression to metastatic disease remains the main cause of cancer death. Yet, the underlying genomic mechanisms driving metastasis remain largely unknown. Here, we present MSK-MET, an integrated pan-cancer cohort of tumor genomic and clinical outcome data from more than 25,000 patients. We analyzed this dataset to identify associations between tumor genomic alterations and patterns of metastatic dissemination across 50 tumor types. We found that chromosomal instability is strongly correlated with metastatic burden in some tumor types, including prostate adenocarcinoma, lung adenocarcinoma and HR-positive breast ductal carcinoma, but not in others, such as colorectal adenocarcinoma, pancreatic adenocarcinoma and high-grade serous ovarian cancer. We also identified specific somatic alterations associated with increased metastatic burden and specific routes of metastatic spread. Our data offer a unique resource for the investigation of the biological basis for metastatic spread and highlight the crucial role of chromosomal instability in cancer progression.

cancer biology↗

bioRxiv2021

MYC controls metastatic heterogeneity in pancreatic cancer

The degree of metastatic disease varies widely amongst cancer patients and impacts clinical outcomes. However, the biological and functional differences that drive the extent of metastasis are poorly understood. We analyzed primary tumors and paired metastases using a multi-fluorescent lineage-labeled mouse model of pancreatic ductal adenocarcinoma (PDAC) - a tumor type where most patients present with metastases. Genomic and transcriptomic analysis revealed an association between metastatic burden and gene amplification or transcriptional upregulation of MYC and its downstream targets. Functional experiments showed that MYC promotes metastasis by recruiting tumor associated macrophages (TAMs), leading to greater bloodstream intravasation. Consistent with these findings, metastatic progression in human PDAC was associated with activation of MYC signaling pathways and enrichment for MYC amplifications specifically in metastatic patients. Collectively, these results implicate MYC activity as a major determinant of metastatic burden in advanced PDAC.

cancer biology↗