Biology subjects

Koche, R. P.

Publications and source records attributed to Koche, R. P..

At least 19 recordsLinked to original sources

bioRxiv2026

Timed binary WNT activity is sufficient to model the early human neural axis

The vertebrate nervous system develops into functionally distinct domains along the body axis[1]. Developmental studies implicate multiple signaling pathways in coordinating this process, but the order of specification events, sufficiency of individual signals, and molecular basis of competency remain unclear [2,3], leaving the field without a unified model of neuraxis patterning. Enteric nervous system (ENS) and midbrain dopaminergic (mDA) progenitors have reached clinical and preclinical testing [4-11], yet neither is produced at full purity, and yields vary markedly across human pluripotent stem cell (hPSC) lines, reflecting incomplete control of axial identity. Here we use single-cell genetic fate mapping to demonstrate that hPSCs can commit to anterior or posterior cranial fate within the first 24 hours of differentiation, before neural induction. Building on this finding, we show that the duration of a single saturating WNT pulse is sufficient to specify three 'first-order' domains: anterior cranial (0 h), posterior cranial (24 h) and trunk (48 h), each pre-neural and competent for CNS, neural crest and epidermal lineages. Nested 'second-order' domains are specified within first-order domains in response to WNT, FGF, and retinoic acid. Mechanistically, an anterior gene regulatory network repressing WNT targets is irreversibly shut down during the transition to posterior identities. Applying this binary WNT patterning logic, we generate mDA neurons and ENS progenitors robustly across hPSC lines and establish an assembloid model combining first-order progenitors to produce all major regions of the nervous system.

developmental biology↗

bioRxiv2026

Quantitative and sensitive neuroblastoma minimal residual disease detection using extrachromosomal DNA (ecDNA) breakpoints

Sensitive detection of minimal residual disease (MRD) remains a major unmet need in high-risk neuroblastoma. MYCN amplification, a hallmark of high-risk disease, typically occurs on extrachromosomal DNA (ecDNA), but the potential of ecDNA-associated genomic rearrangements for individualized MRD monitoring has not been fully exploited. Here, we applied neuroblastoma-specific hybrid capture-based panel sequencing to identify patient-unique breakpoints within MYCN amplicons, and used Circle-seq and Nanopore sequencing to resolve the extrachromosomal amplicon structure in representative samples. Analysis of 8 neuroblastoma cell lines and 22 primary tumors identified 69 tumor-specific breakpoints. Those selected for assay development were validated by breakpoint-specific PCR and Sanger sequencing. Breakpoints detected in primary tumors remained detectable at relapse, supporting their stability as MRD markers. Breakpoint-specific real-time quantitative PCR and droplet digital PCR detected these junctions in bone marrow aspirates with high specificity and reached sensitivities down to a tumor DNA fraction of 10^-6. We applied this approach to 53 serial bone marrow aspirates from 14 patients with high-risk neuroblastoma to monitor MRD dynamics, resolving treatment response and molecular persistence. In six samples, breakpoint-positive DNA was detected in bone marrow that was negative by conventional cytology and immunocytology, highlighting the added value of molecular monitoring. Together, these findings establish ecDNA breakpoint-based detection as a strategy for MRD assessment in neuroblastoma, that is, in principle, applicable to any ecDNA-amplified oncogene.

cancer biology↗

bioRxiv2026

TET2 and TP53 Mutations Cooperatively Modulate the Response to Inflammation to Promote Leukemic Transformation

TET2 is a commonly mutated gene in hematologic malignancies, including as an initiating event in clonal hematopoiesis (CH). Its mutation alters hematopoietic self-renewal, differentiation, and systemic inflammation responses. TP53 mutations co-occur with TET2 mutations and are also observed in patients with high-risk clonal hematopoiesis and hematologic malignancies. Using a murine model, we found that HSPCs with both mutations initially promoted a myeloproliferative phenotype. Over time these double mutant HSPCs acquire additional genomic alternations, leading to disease progression to acute leukemias including B-ALL. We observed enhanced inflammatory signatures at transformation and identified NLRP1 as a target of TP53 activation. Decreased response to an inflammatory cell death pathway in the setting of TP53 mutation allows cells to tolerate inflammatory stress. This pathway also modifies response to chemotherapies that induce protein translational stalling. Our results identify a hematopoietic stem cell stress response pathway with implications on adaptation to inflammation and chemotherapy tolerance. SignificanceTET2 and TP53 mutations co-operate leading to advanced hematologic malignancy. TET2 mutations promote an inflammatory environment and TP53 mutation supports tolerance to this inflammatory stress.

cancer biology↗

bioRxiv2025

Extracellular matrix regulates lineage plasticity in prostate cancer through YAP/TEAD

Treatment-related neuroendocrine prostate cancer (NEPC) is an increasingly frequent mechanism of resistance to androgen receptor pathway inhibitor (ARPI) therapy in prostate adenocarcinoma (PRAD). This lineage transition is dependent on upregulation of the NE-specifying transcription factor ASCL1, typically in a genetic background of RB1 and TP53 loss. Here we identify extracellular matrix-integrin-YAP1/TEAD signaling as a critical brake on NEPC lineage transition. Deletion of Itgb1, the shared B1 subunit required for collagen and laminin-mediated integrin activation, is sufficient to induce ASCL1 and NE lineage gene expression, by activating LATS1/2 kinases with subsequent inactivation of YAP1/TEAD signaling. Conversely, restoration of YAP1/TEAD signaling by pharmacological LATS1/2 inhibition, or by expression of constitutively active YAP1/TAZ mutants, prevents or reverts NEPC lineage transition. NOTCH and AR cooperate with YAP/TEAD to repress ASCL1, such that combined inhibition leads to complete reprograming of PRAD into NEPC in vitro, providing a dynamic platform to dissect the molecular events responsible for lineage transition over time. We find that lineage transition is accompanied by a redistribution of FOXA1 and TEAD cistromes from PRAD to NEPC-specific enhancers and requires the pioneering activity of FOXA1. Thus, extracellular matrix/integrin signaling in the PRAD tumor microenvironment restrains NE lineage plasticity, highlighting a potential path for pharmacological inhibitors in modulating treatment-induced lineage change.

cancer biology↗

bioRxiv2025

The regenerative potential of adult Nestin+ cerebellar astroglia is limited compared to in neonates

There is a crucial need for strategies that stimulate repair in the adult brain. The neonatal mouse cerebellum can regenerate via the adaptive reprogramming of nestin (Nes)-expressing progenitors (NEPs). However, analysis of Nes+ cells of the adult cerebellum is limited. Using reporter lines and genetic inducible fate mapping, we show that adult Nes+ cells are mainly Bergmann glia (Nes+ Bg) that have in vitro sphere-forming ability. Following injury, Nes+ Bg increase in number due to upregulation of Nes in Hopx-expressing Bg, but the cells exhibit limited regeneration. ATAC-seq of Nes+ Bg reveals that silencing of developmental genes compared to neonatal NEPs contributes to the impaired regeneration. Activating sonic hedgehog signalling augments the number of Nes+ Bg after injury but not neurogenesis, showing additional cues are required. Our results demonstrate an age-dependent decline in the regenerative potential of NEPs and highlight Nes+ Bg as potential injury-responsive cells that could facilitate regeneration.

developmental biology↗

bioRxiv2025

L1CAM signaling through planar cell polarity generates SOX2+ metastatic progenitors in lung adenocarcinoma

Plasticity transitions during carcinoma progression generate fetal-like progenitor states with metastatic capacity. How these progenitors emerge and persist during tumor progression remains unknown. Here, we elucidate a process that drives the emergence of SOX2+ metastatic progenitors in lung adenocarcinomas (LUAD). LUAD cells in the tumor invasive front and distant metastases express the cell adhesion molecule L1CAM, a marker of regenerative epithelial progenitors and a mediator of cell-basement membrane and cell-cell interactions. L1CAM-mediated adhesion to perivascular basement membrane is known to stimulate the proliferation of extravasated micrometastatic cells. We now identify a distinct and broader role of L1CAM as promoter of the SOX2+ LUAD progenitor state. We show that L1CAM at cell-cell interfaces promotes the assembly of the planar cell polarity (PCP) complex in metastatic LUAD progenitors. L1CAM-dependent PCP acting through a non-canonical WNT signaling activates c-Jun, which cooperates with the chromatin remodeling factor CHD1 to drive SOX2 expression and metastatic activity. This axis sustains the tumor-initiating and regenerative capacity of LUAD progenitor cells. By illuminating the role of L1CAM and PCP signaling in the generation of SOX2+ LUAD progenitors, our findings identify potential new targets to treat metastatic cancer.

cancer biology↗

bioRxiv2025

Sequential transcriptional gates in the thalamo-cortical circuit coordinate memory stabilization

The molecular mechanisms that enable memories to persist over long time-scales from days to weeks and months are still poorly understood. To develop insights we created a behavioral task where, by varying the frequency of learned associations, mice formed multiple memories but only consolidated some, while forgetting others, over the span of weeks. We then monitored circuit-specific molecular programs that diverge between consolidated and forgotten memories. We identified multiple distinct waves of transcription, i.e., cellular macrostates, specifically in the thalamo-cortical circuit, that defined memory persistence. Notably, a small set of transcriptional regulators orchestrated broad molecular programs that enabled entry into these macrostates. Targeted CRISPR-knockout studies revealed that while these transcriptional regulators had no effects on memory formation, they had prominent, causal, and strikingly time-dependent roles in memory stabilization. In particular, the calmodulin-dependent transcription factor Camta1 was required for initial memory maintenance over days, while Tcf4 and the histone methyl-transferase Ash1l were required later to maintain memory over weeks. These results identify a critical Camta1-Tcf4-Ash1l thalamo-cortical transcriptional cascade required for memory stabilization, and puts forth a model where the sequential, multi-step, recruitment of circuit-specific transcriptional programs enable memory maintenance over progressively longer time-scales.

neuroscience↗

bioRxiv2025

Oncogene Silencing via ecDNA Micronucleation

Extrachromosomal DNA (ecDNA) contributes to cancer genome instability by enabling high-copy oncogene amplification, intratumoural heterogeneity and rapid genetic change. Micronuclei (MN) are frequently observed in chromosomally unstable cancers, yet their origins and relevance in ecDNA-driven tumours remain incompletely understood. Here, we investigate the relationship between ecDNA segregation errors and MN formation. We find that ecDNA frequently localizes to MN and represents a prominent source of MN content in ecDNA-positive cancer cells. Mitotic clustering of oncogene-bearing ecDNAs is associated with asymmetric inheritance and mis-segregation into MN. Transfer of oncogenes from ecDNA into MN is accompanied by reduced transcriptional output. Single-MN sequencing shows that individual MN are enriched for ecDNA to a degree that exceeds expectations from stochastic mis-segregation and that in MN oncogenes originating from multiple distinct ecDNAs coalesce. Using live-cell imaging, we observe that cells inheriting ecDNA-positive MN show limited proliferative capacity and an increased likelihood of cell death. In neuroblastoma patients with MYCN-amplified ecDNA, higher frequencies of ecDNA-positive MN at diagnosis are associated with improved event-free and overall survival. Together, these findings link ecDNA mis-segregation to MN formation and reduced cellular fitness, suggesting that ecDNA-positive MN may reflect a state of impaired oncogenic ecDNA function with potential relevance for clinical outcome.

cancer biology↗

bioRxiv2025

Enhanced FLI1 accessibility mediates STAG2-mutant leukemogenesis

Chromatin architecture governs transcriptional output and cell identity; however, how its disruption promotes leukemic transformation remains incompletely understood. Here, we show that loss of the cohesin subunit STAG2 creates a hyper-accessible chromatin landscape that amplifies FLI1 activity and extends its binding to ectopic loci. Using multi-omic analyses in human AML samples, cell lines, and mouse models, we identify chromatin-dependent co-occupancy of FLI1 and Menin, accompanied by amplification of Menin occupancy at non-canonical loci beyond its HOXA/MEIS1 targets. Therapeutically, the aberrant expansion of Menin binding drives an altered response towards Revumenib and activates interferon response pathways, creating a therapeutic vulnerability to Menin inhibition. Functionally, STAG2/NPM1c co-mutation drives a stem cell-like immunophenotype and a fully penetrant leukemia in vivo. Collectively, these findings define a model in which cohesin loss rewires transcription factor occupancy to amplify oncogenic chromatin programs and expose context-specific therapeutic dependencies.

cancer biology↗

bioRxiv2025

Loss of tumor cell MHC Class II drives insensitivity of BRAF-mutant anaplastic thyroid cancers to MAPK inhibitors

Cancer cells present neoantigens dominantly through MHC class I (MHCI) to drive tumor rejection through cytotoxic CD8+ T-cells. There is growing recognition that a subset of tumors express MHC class II (MHCII), causing recognition of antigens by TCRs of CD4+ T-cells that contribute to the anti-tumor response. We find that mouse BrafV600E-driven anaplastic thyroid cancers (ATC) respond markedly to the RAF + MEK inhibitors dabrafenib and trametinib (dab/tram) and that this is associated with upregulation of MhcII in cancer cells and increased CD4+ T-cell infiltration. A subset of recurrent tumors lose MhcII expression due to silencing of Ciita, the master transcriptional regulator of MhcII, despite preserved interferon gamma signal transduction, which can be rescued by EZH2 inhibition. Orthotopically-implanted Ciita-/- and H2-Ab1-/- ATC cells into immune competent mice become unresponsive to the MAPK inhibitors. Moreover, depletion of CD4+, but not CD8+ T-cells, also abrogates response to dab/tram. These findings implicate MHCII-driven CD4+ T cell activation as a key determinant of the response of Braf-mutant ATCs to MAPK inhibition.

cancer biology↗

bioRxiv2024

An increase in reactive oxygen species underlies neonatal cerebellum repair

The neonatal mouse cerebellum shows remarkable regenerative potential upon injury at birth, wherein a subset of Nestin-expressing progenitors (NEPs) undergoes adaptive reprogramming to replenish granule cell progenitors that die. Here, we investigate how the microenvironment of the injured cerebellum changes upon injury and contributes to the regenerative potential of normally gliogenic-NEPs and their adaptive reprogramming. Single cell transcriptomic and bulk chromatin accessibility analyses of the NEPs from injured neonatal cerebella compared to controls show a temporary increase in cellular processes involved in responding to reactive oxygen species (ROS), a known damage-associated molecular pattern. Analysis of ROS levels in cerebellar tissue confirm a transient increased one day after injury at postanal day 1, overlapping with the peak cell death in the cerebellum. In a transgenic mouse line that ubiquitously overexpresses human mitochondrial catalase (mCAT), ROS is reduced 1 day after injury to the granule cell progenitors, and we demonstrate that several steps in the regenerative process of NEPs are curtailed leading to reduced cerebellar growth. We also provide preliminary evidence that microglia are involved in one step of adaptive reprogramming by regulating NEP replenishment of the granule cell precursors. Collectively, our results highlight that changes in the tissue microenvironment regulate multiple steps in adaptative reprogramming of NEPs upon death of cerebellar granule cell progenitors at birth, highlighting the instructive roles of microenvironmental signals during regeneration of the neonatal brain.

developmental biology↗

bioRxiv2024

Mutant IDH uncouples p53 from target gene regulation to disable tumor suppression

p53 prevents tumor initiation and progression via transcriptional regulation of target gene networks. Here, we find that cancer-associated mutations in isocitrate dehydrogenase (IDH) can uncouple p53 activity from tumor suppression by perturbing chromatin states that determine target gene expression. Mutant IDH impairs tumor regressions and promotes the outgrowth of cancer cells with transcriptionally active, wild-type p53 in a mouse model of liver cancer where restoration of p53 activity results in tumor clearance. Mutant IDH alters p53 target gene expression through the oncometabolite 2-hydroxyglutarate (2-HG), an inhibitor of alpha-ketoglutarate (KG)-dependent chromatin remodeling enzymes, without preventing p53 accumulation or global genomic binding. Rather, mutant IDH alters chromatin accessibility landscapes that dictate target gene expression, resulting in disabled upregulation of targets that execute tumor suppression. Specifically, mutant IDH disrupts the expression of pro-apoptotic p53 targets that enable p53-dependent tumor regressions, including the death ligand receptor Fas. Pharmacological inhibition of mutant IDH in TP53 wild-type cholangiocarcinoma cells, a tumor type where p53 and IDH mutations are mutually exclusive, potentiates p53 target gene expression and sensitizes cells to Fas ligand and chemotherapy-induced apoptosis. Therefore, we implicate the disruption of p53 target gene regulation as a reversible, oncogenic feature of cancer-associated IDH mutations. SIGNIFICANCEWe find that chromatin states altered by cancer-associated IDH mutations intersect with transcriptional regulation of p53 target genes. This reversible interaction may represent a strategy to reinvigorate latent tumor suppression in IDH mutant, p53 wild-type tumors.

cancer biology↗

bioRxiv2024

Aging limits stemness and tumorigenesis in the lung by reprogramming iron homeostasis

Aging is associated with a decline in the number and fitness of adult stem cells1-4. Aging-associated loss of stemness is posited to suppress tumorigenesis5,6, but this hypothesis has not been tested in vivo. Here, using physiologically aged autochthonous genetically engineered mouse models and primary cells7,8, we demonstrate aging suppresses lung cancer initiation and progression by degrading stemness of the alveolar cell of origin. This phenotype is underpinned by aging-associated induction of the transcription factor NUPR1 and its downstream target lipocalin-2 in the cell of origin in mice and humans, leading to a functional iron insufficiency in the aged cells. Genetic inactivation of the NUPR1--lipocalin-2 axis or iron supplementation rescue stemness and promote tumorigenic potential of aged alveolar cells. Conversely, targeting the NUPR1-- lipocalin-2 axis is detrimental to young alveolar cells via induction of ferroptosis. We find that aging-associated DNA hypomethylation at specific enhancer sites associates with elevated NUPR1 expression, which is recapitulated in young alveolar cells by inhibition of DNA methylation. We uncover that aging drives a functional iron insufficiency, which leads to loss of stemness and tumorigenesis, but promotes resistance to ferroptosis. These findings have significant implications for the therapeutic modulation of cellular iron homeostasis in regenerative medicine and in cancer prevention. Furthermore, our findings are consistent with a model whereby most human cancers initiate in young individuals, revealing a critical window for such cancer prevention efforts.

cancer biology↗

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

Mechanical confinement governs phenotypic plasticity in melanoma

Phenotype switching is a form of cellular plasticity in which cancer cells reversibly move between two opposite extremes - proliferative versus invasive states. While it has long been hypothesised that such switching is triggered by external cues, the identity of these cues has remained elusive. Here, we demonstrate that mechanical confinement mediates phenotype switching through chromatin remodelling. Using a zebrafish model of melanoma coupled with human samples, we profiled tumor cells at the interface between the tumor and surrounding microenvironment. Morphological analysis of these rare cells showed flattened, elliptical nuclei suggestive of mechanical confinement by adjacent tissue. Spatial and single-cell transcriptomics demonstrated that the interface cells adopted a gene program of neuronal invasion, including acquisition of an acetylated tubulin cage that protects the nucleus during migration. We identified the DNA-bending protein HMGB2 as a confinement-induced mediator of the neuronal state. HMGB2 is upregulated in confined cells, and quantitative modelling revealed that confinement prolongs contact time between HMGB2 and chromatin, leading to changes in chromatin configuration that favor the neuronal phenotype. Genetic disruption of HMGB2 showed that it regulates the trade-off between proliferative and invasive states, in which confined HMGB2high tumor cells are less proliferative but more drug resistant. Our results implicate the mechanical microenvironment as a mechanism driving phenotype switching in melanoma.

cancer biology↗

bioRxiv2023

Decoil: Reconstructing extrachromosomal DNA structural heterogeneity from long-read sequencing data

Circular extrachromosomal DNA (ecDNA) is a form of oncogene amplification found across cancer types and associated with poor outcome in patients. EcDNA can be structurally complex and contain rearranged DNA sequences derived from multiple chromosome locations. As the structure of ecDNA can impact oncogene regulation and may indicate mechanisms of its formation, disentangling it at high resolution from sequencing data is essential. Even though methods have been developed to identify and reconstruct ecDNA in cancer genome sequencing, it remains challenging to resolve complex ecDNA structures, in particular amplicons with shared genomic footprints. We here introduce Decoil, a computational method which combines a breakpoint-graph approach with LASSO regression to reconstruct complex ecDNA and deconvolve co-occurring ecDNA elements with overlapping genomic footprints from long-read nanopore sequencing. Decoil outperforms de-novo assembly and alignment-based methods in simulated longread sequencing data for both simple and complex ecDNAs. Applying Decoil on whole genome sequencing data uncovered different ecDNA topologies and explored ecDNA structure heterogeneity in neuroblastoma tumors and cell lines, indicating that this method may improve ecDNA structural analyzes in cancer.

cancer biology↗

bioRxiv2023

Intercellular extrachromosomal DNA copy number heterogeneity drives cancer cell state diversity

Neuroblastoma is characterised by extensive inter- and intra-tumour genetic heterogeneity and varying clinical outcomes. One possible driver for this heterogeneity are extrachromosomal DNAs (ecDNA), which segregate independently to the daughter cells during cell division and can lead to rapid amplification of oncogenes. While ecDNA-mediated oncogene amplification has been shown to be associated with poor prognosis in many cancer entities, the effects of ecDNA copy number heterogeneity on intermediate phenotypes are still poorly understood. Here, we leverage DNA and RNA sequencing data from the same single cells in cell lines and neuroblastoma patients to investigate these effects. We utilise ecDNA amplicon structures to determine precise ecDNA copy numbers and reveal extensive intercellular ecDNA copy number heterogeneity. We further provide direct evidence for the effects of this heterogeneity on gene expression of cargo genes, including MYCN and its downstream targets, and the overall transcriptional state of neuroblastoma cells. These results highlight the potential for rapid adaptability of cellular states within a tumour cell population mediated by ecDNA copy number, emphasising the need for ecDNA-specific treatment strategies to tackle tumour formation and adaptation.

cancer biology↗

bioRxiv2022

Epigenetic reprogramming of melanoma cell state through fatty acid β-oxidation and Toll like receptor 4 signaling

Tumor cells respond to a host of factors from the local microenvironment. Microenvironmental fatty acids can be used by melanoma cells for fuel, but their effects on transcription and epigenetics are still unclear. Here, we show that the fatty acid {beta}-oxidation (FAO) pathway integrates signaling and epigenetics to drive melanoma progression. Using transgenic zebrafish and human cell lines, we find that octanoate, a medium-chain fatty acid, increases tumorigenesis. Octanoate is metabolized via the FAO/ACLY axis into acetyl-CoA, leading to increased histone acetylation. Transcriptomic and epigenetic analyses demonstrate a convergence of inflammatory gene signatures in octanoate-treated melanoma cells. This signature is mediated by TLR4/MyD88 signaling, which is activated by saturated fatty acids like octanoate. Genetic inactivation of either FAO enzymes or TLR4/MyD88 inhibits alterations in histone acetylation, and rescues octanoate-tumor promoting effects. Together, these data demonstrate clear evidence linking fatty acid metabolism and epigenetics to melanoma pathogenesis through TLR4 signaling.

cancer biology↗