Biology subjects

Zumbo, P.

Publications and source records attributed to Zumbo, P..

At least 19 recordsLinked to original sources

bioRxiv2026

A human developmental and adult brain atlas benchmarks dopaminergic stem cell models and cell therapy candidates

Parkinsons disease (PD) is characterized by the progressive loss of midbrain dopaminergic (mDA) neurons1. Stem cell-derived mDA neurons hold promise for disease modelling2,3 and are currently in clinical trials for cell replacement therapy4-6. However, systematic benchmarking has been limited by the lack of a unified high-resolution reference and methods that quantify incomplete or mixed lineage specification in vitro7. We establish a single-cell and spatial atlas of the human developing diencephalon-midbrain-hindbrain axis resolving 93 cell subtypes, including 39 lacking prior single-cell characterization and 4 entirely novel populations. Using this atlas as a reference, we integrate 19 hPSC-derived mDA datasets, both published2,8-25 and unpublished, to build the Human Dopaminergic Neural Atlas (HDNA) spanning 2D, 3D, and graft models, including those used in clinical trials. To classify cells and quantify lineage fidelity, we develop CapybaraBrain, a marker-driven non-negative decomposition framework that assigns each cell continuous identity scores across all 93 developmental programs, enabling systematic discrimination of discrete, transitioning, and cross-lineage hybrid states26. We uncover a pervasive landscape of off-target populations reflecting relaxed transcriptional boundaries in vitro, including a previously unrecognized TH-PITX2 midbrain neuronal population, and we validate atlas-predicted latent lineage plasticity through inducible genetic fate mapping in mouse models. We further define maturation-associated transcriptional programs by harmonizing adult mDA subtype atlases, revealing that dopaminergic identity and maturation are partially decoupled across protocols. Finally, projecting PD patient-derived tri-cultures onto the HDNA uncovers genotype- and cell-type-specific transcriptional dysregulation. Together, these integrated atlases and computational framework establish a unified standard for benchmarking differentiation fidelity, exposing off-target states, and guiding next-generation PD models and cell therapies.

Developmental Biology↗

bioRxiv2026

Hypoxia-activated scleraxis a mediates epicardial progenitor differentiation into a unique cardiac perivascular cell type

The epicardium is a crucial source of progenitor cells and paracrine signals that support heart development and regeneration. However, the molecular mechanisms that guide epicardial cell fate decisions remain incompletely understood. Here, we identify the transcription factor Scleraxis a (encoded by scxa) as a key regulator of epicardial progenitor differentiation in zebrafish. Through single-cell transcriptomics, genetic lineage tracing, and cardiac injury models, we show that scxa is transiently induced in activated epicardial progenitor cells (aEPCs) during both heart regeneration and developmental coronary angiogenesis. scxa+ epicardial cells primarily give rise to a previously uncharacterized cardiac population of perivascular cells marked by col18a1a, molecularly distinct from classical pericytes and vascular smooth muscle cells. We refer to this population as epicardial-derived perivascular mesenchymal cells (Epi-PMCs). These Epi-PMCs closely associate with coronary vessels and may contribute to vascular stabilization and remodeling, potentially through the anti-angiogenic but vessel-stabilizing activity of endostatin derived from collagen XVIII. Loss of scxa increases coronary vessel density. Mechanistically, we identify hypoxia and Hif1a signaling as upstream regulators of scxa, with systemic hypoxia or Hif factor stabilization robustly inducing scxa expression in the epicardium. Together, these findings uncover a hypoxia-responsive Scxa-Col18a1a axis that drives epicardial differentiation toward a vascular-supportive fate, offering new insight into the regulation of coronary vessel development and the regenerative potential of the epicardium.

developmental biology↗

bioRxiv2026

Dynamic Antigen Expression and Intrinsic CTL Resistance in HIV Reservoir Clones

Reservoirs of clonally expanded CD4+ T-cells harboring rebound-competent HIV proviruses persist lifelong during ART. Latency is considered the principal barrier to viral eradication and has resisted pharmacological reversal, yet it appears that sustained immune pressure may still erode reservoirs. Recent advances have yielded glimpses into these exceptionally rare reservoir-harboring cells, implicating intrinsic pro-survival properties in their persistence. Here, we isolate and characterize populations of authentic reservoir clones (ARCs) that robustly proliferate and accumulate while producing infectious virus, without overtly succumbing to viral cytopathic effects. At any given moment, only small fractions of ARCs expressed HIV proteins, a state remarkably unperturbed by potent TCR or mitogenic stimulation. Nevertheless, sustained co-culture with cytotoxic T-lymphocytes (CTL) revealed extensive time-integrated antigenic vulnerability, culling clonal expansion of some ARCs by >90%. Notably, a regulatory T-cell ARC displayed pronounced cell-intrinsic resistance to CTL - a longstanding hypothesis we now directly demonstrate - linked to low oxidative stress and reversed with desferoxamine, a hypoxic stress inducer and FDA-approved therapeutic. Overall, we provide novel insights into the vulnerabilities of reservoir clones to potent, sustained CTL pressure and highlight intrinsic resistance pathways as actionable therapeutic targets, opening opportunities for advancing immune-based HIV cure strategies.

immunology↗

bioRxiv2025

HIV Nef amplifies mechanical heterogeneity to promote immune evasion

Intracellular pathogens must evade cytotoxic immunity to establish persistent infection. Although immune escape is typically viewed through a biochemical lens, the ability of certain pathogens to alter the mechanical properties of infected cells suggests that biophysical mechanisms may also contribute to the process. Here, we show that a subset of CD4+ T cells infected with the human immunodeficiency virus (HIV) resist elimination through a soft phenotype that inhibits killing by mechanosensitive cytotoxic T lymphocytes (CTLs). This phenotype arises from the combined effects of the HIV virulence factor Nef, which remodels the actin cytoskeleton, and intrinsic heterogeneity in the basal cytoskeletal properties of infected T cells. Pharmacological or genetic perturbations that reverse Nef signaling to the cytoskeleton or that stiffen the filamentous-actin cortex sensitize infected cells to CTL-mediated lysis. Taken together, these findings define a novel, biophysical paradigm of immune evasion with implications for HIV cure strategies.

immunology↗

bioRxiv2025

Pre-division TCF1 drop determines long-term CD8 T cell fates

T Cell Factor 1 (TCF1) is a master transcription factor controlling T cell development and peripheral T cell differentiation during infection, cancer, and autoimmunity. TCF1 is highly expressed in naive CD8 T cells but must be downregulated as T cells proliferate to become effectors. If and how TCF1 plays a role during T cell priming prior to cell cycle entry is unknown. Surprisingly, we found that TCF1 expression is rapidly downregulated within hours after antigen encounter in both murine and human CD8 T cells, even before T cells enter cell cycle. TCF1 then rebounds to high levels upon cell cycle entry, ultimately declining again with proliferation and effector differentiation. This rapid pre-division drop and rebound occurs in diverse settings, including infection and cancer. The magnitude of the pre-division TCF1 drop is modulated by TCR signal strength and inflammatory cytokines and strikingly, regulates long-term effector and memory fates. Paired transcriptomic and epigenetic analyses revealed that TCF1-regulated chromatin regions were remodeled within hours following antigen encounter, activating effector and inflammatory cytokine signaling modules and poising T cells for effector differentiation. Remarkably, transient siRNA-mediated TCF1 downmodulation during the pre-division priming phase was sufficient to induce long-term population effector skewing. We have uncovered a novel mechanism whereby pre-division dynamic TCF1 regulation determines long-term CD8 T cell fate commitment, potentially serving as a critical checkpoint regulating T cell responses in infection, cancer, and autoimmunity.

immunology↗

bioRxiv2025

Lysosome-dependent nutrient scavenging underliesstress adaptation during epithelial-to-mesenchymaltransition

Metastatic cancer cells invade tissue, overcome nutrient stress, and survive transit to distant sites. Many of the mechanisms that support these processes are incompatible with proliferation. This study defines cellular transition states in breast epithelial cells undergoing epithelial-mesenchymal transition (EMT) driven by ERK2 and TGF-{beta} signaling. EMT triggers robust endolysosomal system upregulation and metabolic adaptations that balance proliferative and invasive states. Surprisingly, invasive cells rely on scavenging via lysosomes and macropinocytosis to acquire amino acids, rather than plasma membrane transport, even in nutrient-rich conditions. Macropinocytosis increases intracellular amino acid storage, promoting survival during amino acid deprivation. This metabolic shift depends on c-MYC downregulation, an early EMT event. Reintroducing c-MYC suppresses the metabolic switch, endolysosomal induction, macropinocytosis, and the proliferation-to-migration transition. These findings reveal how cells dynamically balance proliferation and invasion, offering insights into transition states difficult to capture in models of breast cancer metastasis.

cell biology↗

bioRxiv2025

CCAAT-enhancer binding protein delta functions as a tumor suppressor gene in acute myeloid leukemia

There is a continued need for identification of novel disease drivers of acute myeloid leukemia as many patients experience relapse and have poor clinical outcomes. Analyses from our study and publicly available datasets predicted CEBPD as a novel tumor suppressor gene in acute myeloid leukemia. Consistent with the analyses, CEBPD knockdown experiments showed activation of MAPK signaling with concomitant increase in cell growth rate, while upregulation experiments suggested induction of myeloid differentiation marker CD14 expression in AML cell lines OCI-AML2 and OCI-AML5. Consistent with a previous report, our genomics analyses and azacytidine treatment experiments suggested a role for DNA methylation in downregulation of CEBPD expression during AML pathogenesis. Altogether, our results provide experimental evidence for a tumor suppressor function of CEBPD in AML.

cancer biology↗

bioRxiv2025

tRNA modifications are required for stress granule formation and melanoma metastasis

Metastasis is the leading cause of cancer related deaths, however therapies specifically targeting metastasis are lacking and remain a dire therapeutic need in the clinic. Metastasis is a highly inefficient process that is inhibited by extracellular stress. Therefore, metastasizing cells that ultimately survive and successfully colonize distant organs must undergo molecular rewiring to mitigate stress. Wobble uridine modifications, especially 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), have been implicated in stress response and poor prognosis of cancer patients. We use a patient derived xenograft (PDX) model of melanoma metastasis to study the role of the mcm5s2U34 modification in the stress response of metastasizing cells. We find that upon depletion of elongator acetyltransferase complex subunit 1 (ELP1)-- a component of the mcm5s2U34 pathway on [Formula], and [Formula] --codon-biased translation, migration, invasion, and metastatic burden in vivo is reduced. Further, we observe that stress granule components are enriched in a subset of codon-biased genes that are exclusively upregulated at the protein level in metastatic nodules compared to the primary tumor in our PDX model. Additionally, upon knockdown of ELP1, stress granule components have decreased protein expression with no significant change to their mRNA levels. Efficient translation, mediated by the carboxy-methylation arm of the mcm5s2U34 modification, is required for metastasizing cancer cells to withstand stress via stress granule formation and increase survival throughout the metastatic cascade. This makes the mcm5s2U34 machinery a potentially actionable therapeutic target, specific to metastatic disease.

cancer biology↗

bioRxiv2025

Molecular dynamics driving phenotypic divergence among KRAS mutants in pancreatic tumorigenesis

Inflammation in the pancreas drives acinar-to-ductal metaplasia (ADM), a progenitor-like state that can be hijacked by mutant Kras in the formation of pancreatic cancer (PDAC). How these cell fate decisions vary according to KRAS mutation remains poorly understood. To define mutation-specific lineage reversion and tumor initiation, we implement novel Ptf1a-TdTomato mice and multiple KRAS mutants across an array of genetic, pharmacologic, and inflammatory perturbations in vivo. Whereas KRASG12D co-opts injury to enable lineage reversion, enhancer reprogramming, and tumor initiation, KRASG12R/V can initiate but not sustain dedifferentiated and neoplastic transcriptional and epigenetic programs. We find the KRASG12R/V defects consist of a failure to invoke robust EGFR signaling and activate Rac1/Vav1, with constitutive Akt activation in vivo sufficient to rescue the tumorigenic potential of KRASG12R. As the marked heterogeneity among KRAS variants begins early in tumorigenesis, these data are crucial to understanding mutation-specific oncogenic trajectories and directing the implementation of KRAS-directed therapeutics. SIGNIFICANCEDefining how KRAS mutants drive distinct outcomes in human pancreatic cancer is critical for developing allele-specific therapeutic approaches. This study unveils a hierarchy among KRASG12D, KRASG12V, and KRASG12R to drive tumor initiation, owing to heterogeneous activation of EGFR, PI3K/AKT, and RAC1 signaling, thus revealing mutation-specific evolutionary paths in pancreatic tumorigenesis.

cancer biology↗

bioRxiv2024

Multi-Omic Atlas reveals cytotoxic phenotype and ROS-linked metabolic quiescence as key features of CTL-resistant HIV-infected CD4+ T-cells

Cytotoxic T-lymphocytes (CTL) exert sustained pressure on reservoirs of HIV-infected cells that persist through years of antiretroviral therapy (ART). This selects for latently infected cells, but also potentially for cells that express HIV but possess intrinsic CTL resistance. We demonstrate that such resistance exists in HIV-infected CD4+ T-cells that survive rigorous CTL attack and map CTL susceptibility to cell identities and states defined by single-cell multi-omics and functional metabolic profiling. Cytotoxic CD4+ T-cells were prominently overrepresented amongst survivors, as were cells with quiescent metabolic profiles and low levels of reactive oxygen species (ROS) production. The induction of ROS production by treatment with deferoxamine sensitized these cells to CTL-mediated elimination. Reservoir-harboring cells from clinical samples share the above transcriptional features, being enriched for quiescent states. Our results provide an atlas for elucidating features of CTL resistance in HIV reservoirs, and identify oxidative stress as a therapeutic target to facilitate reservoir elimination.

immunology↗

bioRxiv2024

An IFN-STAT1-CYBB Axis Defines Protective Plasmacytoid DC to Neutrophil Crosstalk During Aspergillus fumigatus Infection.

Aspergillus fumigatus is the most common cause of invasive aspergillosis (IA), a devastating infection in immunocompromised patients. Plasmacytoid dendritic cells (pDCs) regulate host defense against IA by enhancing neutrophil antifungal properties in the lung. Here, we define the pDC activation trajectory during A. fumigatus infection and the molecular events that underlie the protective pDC - neutrophil crosstalk. Fungus-induced pDC activation begins after bone marrow egress and results in pDC-dependent regulation of lung type I and type III IFN levels. These pDC-derived products act on type I and type III IFN receptor-expressing neutrophils and control neutrophil fungicidal activity and reactive oxygen species production via STAT1 signaling in a cell-intrinsic manner. Mechanistically, neutrophil STAT1 signaling regulates the transcription and expression of Cybb, which encodes one of five NADPH oxidase subunits. Thus, pDCs regulate neutrophil-dependent immunity against inhaled molds by controlling the local expression of a subunit required for NADPH oxidase assembly and activity in the lung. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/620079v2_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1394ccforg.highwire.dtl.DTLVardef@e1bb13org.highwire.dtl.DTLVardef@1c0f991org.highwire.dtl.DTLVardef@40d5db_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

The EZH2 inhibitor tazemetostat mitigates HIV immune evasion, reduces reservoir formation, and promotes durable CD8+ T-cell revitalization

Persistent HIV reservoirs in CD4 T-cells pose a barrier to curing HIV infection. We identified overexpression of enhancer of zeste homolog 2 (EZH2) in HIV-infected CD4 T- cells that survive cytotoxic T lymphocyte (CTL) exposure, suggesting a mechanism of CTL resistance. Inhibition of EZH2 with the FDA-approved drug tazemetostat increased surface expression of major histocompatibility complex class I (MHC-I) on CD4 T-cells, counterbalancing HIV Nef-mediated MHC-I downregulation. This improved CTL-mediated elimination of HIV-infected cells and suppressed viral replication in vitro. In a participant-derived xenograft mouse model, tazemetostat elevated MHC-I and the pro-apoptotic protein BIM in CD4 T-cells, facilitating CD8 T-cell-mediated reductions of HIV reservoir seeding. Additionally, tazemetostat promoted sustained skewing of CD8 T-cells toward less differentiated and exhausted phenotypes. Our findings reveal EZH2 overexpression as a novel mechanism of CTL resistance and support the clinical evaluation of tazemetostat to enhance clearance of HIV reservoirs and improve CD8+ T-cell function.

immunology↗

bioRxiv2024

CAR-engineered lymphocyte persistence is governed by a FAS ligand/FAS auto-regulatory circuit

Chimeric antigen receptor (CAR)-engineered T and NK cells can cause durable remission of B-cell malignancies; however, limited persistence restrains the full potential of these therapies in many patients. The FAS ligand (FAS-L)/FAS pathway governs naturally-occurring lymphocyte homeostasis, yet knowledge of which cells express FAS-L in patients and whether these sources compromise CAR persistence remains incomplete. Here, we constructed a single-cell atlas of diverse cancer types to identify cellular subsets expressing FASLG, the gene encoding FAS-L. We discovered that FASLG is limited primarily to endogenous T cells, NK cells, and CAR-T cells while tumor and stromal cells express minimal FASLG. To establish whether CAR-T/NK cell survival is regulated through FAS-L, we performed competitive fitness assays using lymphocytes modified with or without a FAS dominant negative receptor ({Delta}FAS). Following adoptive transfer, {Delta}FAS-expressing CAR-T and CAR-NK cells became enriched across multiple tissues, a phenomenon that mechanistically was reverted through FASLG knockout. By contrast, FASLG was dispensable for CAR-mediated tumor killing. In multiple models, {Delta}FAS co-expression by CAR-T and CAR-NK enhanced antitumor efficacy compared with CAR cells alone. Together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS-L/FAS auto-regulatory circuit.

immunology↗

bioRxiv2023

Selenocysteine tRNA methylation promotes oxidative stress resistance in melanoma metastasis

Selenocysteine-containing proteins play a central role in redox homeostasis. Their translation is a highly regulated process, dependent upon two tRNASec isodecoders differing by a single 2-O-ribose methylation, called Um34. We characterized FTSJ1 as the Um34 methyltransferase and show that its activity is required for efficient selenocysteine insertion at the UGA stop codon during translation. Specifically, Loss of Um34 leads to ribosomal stalling and decreased UGA recoding. FTSJ1-deficient cells are more sensitive to oxidative stress and have decreased metastatic colonization in xenograft models of melanoma metastasis. We found that FTSJ1 mediates efficient translation of selenoproteins essential for the cellular antioxidant response. Our findings uncover a role for tRNASec Um34 modification in oxidative stress resistance and highlight FTSJ1 as a potential therapeutic target specific for metastatic disease.

cancer biology↗

bioRxiv2023

Intratumoral immune triads are required for adoptive T cell therapy-mediated elimination of solid tumors

Tumor-reactive CD8 T cells found in cancer patients are frequently dysfunctional, unable to halt tumor growth. Adoptive T cell transfer (ACT), the administration of large numbers of in vitro-generated cytolytic tumor-reactive CD8 T cells, is an important cancer immune therapy being pursued. However, a limitation of ACT is that transferred CD8 T cells often rapidly lose effector function, and despite exciting results in certain malignancies, few ACT clinical trials have shown responses in solid tumors. Here, we developed preclinical cancer mouse models to investigate if and how tumor-specific CD4 T cells can be enlisted to overcome CD8 T cell dysfunction in the setting of ACT. In situ confocal microscopy of color-coded cancer cells, tumor-specific CD8 and CD4 T cells, and antigen presenting cells (APC), combined with functional studies, revealed that the spatial positioning and interactions of CD8 and CD4 T cells, but not their numbers, dictates ACT efficacy and anti-tumor responses. We uncover a new role of antigen-specific CD4 T cells in addition to the known requirement for CD4 T cells during priming/activation of naive CD8 T cells. CD4 T cells must co-engage with CD8 T cells and APC cross-presenting CD8-and CD4-tumor antigens during the effector phase, forming a three-cell-cluster (triad), to license CD8 T cell cytotoxicity and mediate cancer cell elimination. Triad formation transcriptionally and epigenetically reprogram CD8 T cells, prevent T cell dysfunction/exhaustion, and ultimately lead to the elimination of large established tumors and confer long-term protection from recurrence. When intratumoral triad formation was disrupted, adoptively transferred CD8 T cells could not be reprogrammed, and tumors progressed despite equal numbers of tumor-infiltrating CD8 and CD4 T cells. Strikingly, the formation of CD4 T cell::CD8 T cell::APC triads in tumors of patients with lung cancers treated with immune checkpoint blockade was associated with clinical responses, but not CD4::APC dyads or overall numbers of CD8 or CD4 T cells, demonstrating the importance of triads in non-ACT settings in humans. Our work uncovers intratumoral triads as a key requirement for anti-tumor immunity and a new role for CD4 T cells in CD8 T cell cytotoxicity and cancer cell eradication.

immunology↗

bioRxiv2023

Cross-species single-cell comparison of systemic and cardiac inflammatory responses after cardiac injury

The immune system coordinates the response to cardiac injury and is known to control regenerative and fibrotic scar outcomes in the heart and subsequent chronic low-grade inflammation associated with heart failure. Here we profiled the inflammatory response to heart injury using single cell transcriptomics to compare and contrast two experimental models with disparate outcomes. We used adult mice, which like humans lack the ability to fully recover and zebrafish which spontaneously regenerate after heart injury. The extracardiac reaction to cardiomyocyte necrosis was also interrogated to assess the specific peripheral tissue and immune cell reaction to chronic stress. Cardiac macrophages are known to play a critical role in determining tissue homeostasis by healing versus scarring. We identified distinct transcriptional clusters of monocytes/macrophages in each species and found analogous pairs in zebrafish and mice. However, the reaction to myocardial injury was largely disparate between mice and zebrafish. The dichotomous response to heart damage between the mammalian and zebrafish monocytes/macrophages may underlie the impaired regenerative process in mice, representing a future therapeutic target.

cell biology↗

bioRxiv2022

Activation of an injury-associated transient progenitor state in the epicardium is required for zebrafish heart regeneration

The epicardium, a mesothelial cell tissue that encompasses vertebrate hearts, supports heart regeneration after injury through paracrine effects and as a source of multipotent progenitors. However, the progenitor state in the adult epicardium has yet to be defined. Through single-cell RNA-sequencing of isolated epicardial cells from uninjured and regenerating adult zebrafish hearts, we defined the epithelial and mesenchymal subsets of the epicardium. We further identified a transiently activated epicardial progenitor cell (aEPC) subpopulation marked by ptx3a and col12a1b expression. Upon cardiac injury, aEPCs emerge from the epithelial epicardium, migrate to enclose the wound, undergo epithelial-mesenchymal transition (EMT), and differentiate into mural cells and pdgfra+hapln1a+ mesenchymal epicardial cells. These EMT and differentiation processes are regulated by the Tgf{beta} pathway. Conditional ablation of aEPCs blocked heart regeneration through reduced Nrg1 expression and mesenchymal cell number. Our findings identify a transient progenitor population of the adult epicardium that is indispensable for heart regeneration and highlight it as a potential target for enhancing cardiac repair.

developmental biology↗

bioRxiv2022

Genetic or transcranial magnetic stimulation of B-RAF-MEK signaling promotes CST axon sprouting and functional regeneration

Facilitating axon regeneration in the injured central nervous system remains a challenging task. RAF-MEK signaling plays an important role in axon elongation during nervous system development. Here we show that activation of B-RAF in mature corticospinal neurons elicited the expression of a discrete set of transcription factors previously implicated in the regeneration of zebrafish optic nerve axons. Genetic activation of B-RAF-MEK signaling promoted robust regeneration and sprouting of corticospinal tract axons after injury. Newly sprouting axon collaterals formed synaptic connections with spinal interneurons, correlating with the recovery of skilled motor function. Seeking a non-invasive way to stimulate axon regeneration, we found that suprathreshold high-frequency repetitive transcranial magnetic stimulation activates the B-RAF canonical effectors MEK1/2 and requires MEK1/2 activity to promote corticospinal axon regeneration and sprouting after injury. These data demonstrate a central role of neuron-intrinsic RAF-MEK signaling in enhancing the growth capacity of mature corticospinal neurons and propose HF-rTMS as a potential therapy for spinal cord injury. One Sentence SummaryGenetic or HF-rTMS-mediated activation of B-RAF- MEK signaling promotes CST axon sprouting and functional regeneration after a spinal cord injury.

neuroscience↗