Biology subjects

Lipka, D. B.

Publications and source records attributed to Lipka, D. B..

12 recordsLinked to original sources

bioRxiv2026

scIRES-seq reveals multimodal molecular heterogeneity in CTCs from small cell cervical cancer

Circulating tumor cells (CTCs) can provide a window into cellular and molecular mechanisms of cancer dissemination. However, the rarity and heterogeneity of CTCs complicate reliable identification and molecular characterization. Here, we introduce scIRES-seq, a framework linking immunophenotype, genetic variation, epigenomic profiles and gene expression within individual cells. In this proof-of-concept study, we coupled scIRES-seq to real-time imaging spectral flow cytometry and applied it to CTCs from a patient with metastatic cervical small cell neuroendocrine carcinoma. Tumor-matched copy-number alterations and targeted somatic variants established CTC identity, supported by DNA methylation profiles concordant with metastatic tissue and transcriptional features consistent with neuroendocrine differentiation. Genetic and DNA methylation analyses identified two largely concordant CTC subpopulations that were not clearly distinguished by their transcriptomes, highlighting the complementary information gained through single-cell multi-omics profiling. These findings demonstrate the feasibility of integrated CTC identification and characterization and provide a framework for investigating relationships between genetic diversity and molecular states in rare, heterogeneous cell populations.

cancer biology↗

bioRxiv2026

Hierarchical classification of hematologic malignancies using epigenetic and genetic information

Molecular testing in hematology requires different assays for disease subgroup identification, risk stratification and selection of appropriate treatment regimens. Yet, molecular tests are not necessarily standardized between diagnostic laboratories, resulting in varying turnaround times and potentially divergent results. To resolve this issue and enable single-assay molecular testing, we have developed a hierarchical classification framework that combines epigenetic and genetic data from whole genome nanopore sequencing (WGNS) with machine learning to determine disease entities, epigenetic subgroups (epitypes) and genetic aberrations in hematopoietic neoplasms. We curated DNA methylation data from 5,420 samples and trained a classifier allowing entity-level diagnostics featuring 21 conditions, including healthy controls, acute and chronic myeloid and lymphoid neoplasms. This classifier was subsequently combined with entity-specific epitype classifiers predicting 44 therapeutically or prognostically relevant states, followed by integration of genetic data. Benchmarking of the combined (epi-)genetic testing strategy using WGNS confirmed high accuracy in the detection of diagnostic groups and risk stratification, and identified diagnosis-defining molecular alterations that were not reported by standard-of-care work-up.

cancer biology↗

bioRxiv2026

Dormancy, not apoptosis, restricts hematopoietic stem cell mutagenesis during aging

Genome instability and mutagenesis are hallmarks of aging, acting as drivers of some age-associated pathologies, including cancer1-3. Somatic cells engage multiple layers of protection against mutagenesis, including detoxification of genotoxic metabolites; repair of DNA damage; and elimination of cells which suffer excessive damage4-7. In this context, the intrinsic apoptotic pathway is engaged in response to activation of the DNA damage response (DDR) and is thought to play a major role in limiting accumulation of mutations, particularly in cells that act as an origin for cancer, such as somatic stem cells8,9. However, the dissection of the relative contribution of different protective mechanisms that restrict mutagenesis in such cells is confounded by the long time frame of experiments; relatively low mutation burden in non-malignant cells; and high variance across individuals due to differences in germ line and environment. Here we employ deep whole-genome sequencing (WGS) combined with extended time-course sampling from a range of experimental mouse models to study mutation acquisition in hematopoietic stem cells (HSCs) during aging. Having validated that murine HSCs recapitulate mutation acquisition patterns observed in aged human HSCs, we made the surprising discovery that apoptosis has a negligible role in restricting mutagenesis. Instead, we found that HSC dormancy inhibits mutagenesis during normal aging, with dormant HSCs from old mice demonstrating a mutation burden akin to their young counterparts. Importantly, breaking HSC dormancy via induction of sterile inflammation led to a dramatic acceleration in mutation rate, demonstrating that non-genotoxic environmental stimuli can modulate genome stability. These findings provide new insights into the correlation between inflammation and both aging and carcinogenesis.

cell biology↗

bioRxiv2025

Oncogenic PTPN11/SHP2 drives immune escape in juvenile myelomonocytic leukemia (JMML) through activation of ectonucleotidase/adenosine signaling

Juvenile myelomonocytic leukemia (JMML) is a myelodysplastic/myeloproliferative neoplasm of early childhood driven by RAS pathway mutations. Allogeneic hematopoietic stem cell transplantation (HSCT) is the therapy of choice for most patients. However, relapse rate is high, in patients with adverse features, frequently noted in PTPN11-mutated JMML, or in patients without evidence of graft-versus-host disease (GvHD). Here we set out to understand the mechanisms associated with oncogenic PTPN11 immune escape. Analyzing primary PTPN11-mutated JMML samples and MxCre;Ptpn11D61Y/+ mice, we observed elevated expression of immune checkpoint molecules, including ectonucleotidases CD39 and CD73 - key mediators of the adenosine pathway - on monocytic and granulocytic leukemic cells. Stimulation with GM-CSF, a central mediator of JMML pathogenesis, induced ectonucleotidases expression on granulocytes and monocytes. In contrast, MEK inhibition downstream of Ptpn11D61Y/+ reduced ectonucleotidases expression. Functionally, Ptpn11D61Y/+-mutated myeloid cells suppressed activation and proliferation of wild-type (WT) T lymphocytes, an effect recapitulated by adenosine and reversed by pharmacological CD39 inhibition with POM-1. In vivo, POM-1 treatment of MxCre;Ptpn11D61Y/+mice presenting with myeloproliferation reduced spleen size and partially restored immune responsiveness. Moreover, POM-1 induced apoptosis in murine Ptpn11D61Y/+ myeloid cells, highlighting a dual therapeutic benefit of CD39 inhibition in JMML. Together, these findings suggest that targeting the adenosine pathway may represent an immunomodulatory approach to enhance T cell-mediated control of JMML, particularly in the context of HSCT and relapse prevention.

cancer biology↗

bioRxiv2024

High interleukin-3 concentrations prime murine myeloid progenitor cells towards granulocytic differentiation

Impaired differentiation a key feature of many hematopoietic malignancies. To decipher the molecular processes underlying malignant transformation, it is important to understand the mechanisms regulating hematopoietic differentiation. Cell lines derived from transformed hematopoietic progenitors or from leukemia patients have proven to be valuable model systems for mechanistic investigations of hematopoiesis. In the present work, we investigated the cell-intrinsic differentiation capacity of the interleukin 3 (IL-3) dependent murine myeloid progenitor cell line 32D. We demonstrated that 32D cells have a cell-intrinsic granulocytic differentiation potential which requires the presence of high IL-3 concentrations in order to induce granulocytic priming. We also show that 32D cells still proliferate at comparable rates but lack granulocytic priming in the presence of intermediate IL-3 concentrations. This phenotypic plasticity is fully reversible and entirely depends on the IL-3 concentrations present in the culture media. RNA-seq analysis revealed that, among other myeloid genes, the expression of Csf3r is strongly induced exclusively in the presence of high IL-3 concentrations, likely explaining the granulocytic differentiation in response to G-CSF treatment. Together, our work provides detailed cellular and molecular insights into the phenotypic plasticity of 32D cells driven by different IL-3 concentrations and suggest culture conditions for different experimental set ups. Key pointsO_LI32D cells represent a murine myeloid cell line with endogenous granulocytic differentiation potential. C_LIO_LI32D cells present molecular and functional plasticity depending on their culture conditions. C_LIO_LIHigh IL-3 culture conditions prime 32D cells towards the granulocytic lineage. C_LIO_LILow IL-3 culture conditions lead to a reversible loss of the granulocytic differentiation potential of 32D cells while fully maintaining their proliferative activity. C_LI

molecular biology↗

bioRxiv2023

Oncogenic RAS-Pathway Activation Drives Oncofetal Reprogramming and Creates Therapeutic Vulnerabilities in Juvenile Myelomonocytic Leukemia

Aberrant fetal gene expression facilitates tumor-specific cellular plasticity by hijacking molecular programs of embryogenesis1. Persistent fetal gene signatures in childhood malignancies are typically explained by their prenatal origins2-6. In contrast, reactivation of fetal gene expression is considered a consequence of oncofetal reprogramming (OFR) in adult malignancies and is associated with aggressive disease7-10. To date, OFR has not been described in the context of childhood malignancies. Here, we performed a comprehensive multi-layered molecular characterization of juvenile myelomonocytic leukemia (JMML) and identified OFR as a hallmark of aggressive JMML. We observed that hematopoietic stem cells (HSCs) aberrantly express mixed developmental programs in JMML. Expression of fetal gene signatures combined with a postnatal epigenetic landscape suggested OFR, which was validated in a JMML mouse model, demonstrating that postnatal activation of RAS signaling is sufficient to induce fetal gene signatures. Integrative analysis identified the fetal HSC maturation marker CD52 as a novel therapeutic target for aggressive JMML. Anti-CD52 treatment depleted human JMML HSCs and disrupted disease propagation in vivo. In summary, this study implicates OFR, defined as postnatal acquisition of fetal transcription signatures, in the pathobiology of a childhood malignancy. We provide evidence for the direct involvement of oncogenic RAS signaling in OFR. Finally, we demonstrate how OFR can be leveraged for the development of novel treatment strategies. Highlights{blacksquare} Epigenomic and transcriptomic landscape of juvenile myelomonocytic leukemia (JMML) in the context of hematopoietic development. {blacksquare}The presence of fetal transcription signatures in childhood malignancies is not indicative of a developmental maturation block. {blacksquare}High-risk JMML is characterized by oncofetal reprogramming of postnatal hematopoietic stem cells (HSCs). {blacksquare}RAS-pathway mutations induce fetal-like gene expression signatures in murine postnatal HSCs. {blacksquare}The fetal maturation marker CD52 is a novel therapeutic target in high-risk JMML.

cancer biology↗

bioRxiv2023

Altered enhancer-promoter interaction leads to MNX1 expression in pediatric acute myeloid leukemia with t(7;12)(q36;p13)

Acute myeloid leukemia (AML) with the t(7;12)(q36;p13) translocation occurs only in very young children and has a poor clinical outcome. The expected oncofusion between breakpoint partners (MNX1 and ETV6) has only been reported in a subset of cases. However, a universal feature is the strong transcript and protein expression of MNX1, a homeobox transcription factor that is normally not expressed in hematopoietic cells. Here, we map the translocation breakpoints on chromosomes 7 and 12 in affected patients to a region proximal to MNX1 and either introns 1 or 2 of ETV6. The frequency of MNX1 overexpression in pediatric AML (n=1556, own and published data) is 2.4% and occurs predominantly in t(7;12)(q36;p13) AML. Chromatin interaction assays in a t(7;12)(q36;p13) iPSC cell line model unravel an enhancer-hijacking event that explains MNX1 overexpression in hematopoietic cells. Our data suggest that enhancer-hijacking is a more common and overlooked mechanism for structural rearrangement-mediated gene activation in AML. Key pointsO_LIExpression analysis of over 1500 pediatric AML samples demonstrates MNX1 expression as a universal feature of t(7;12)(q36;p13) AML as well as in rare cases without t(7;12)(q36;p13) C_LIO_LIMNX1 is activated by an enhancer-hijacking event in t(7;12)(q36;p13) AML and not, as previously postulated, by the creation of a MNX1::ETV6 oncofusion gene. C_LI

cancer biology↗

bioRxiv2022

Dynamic DNA methylation reveals novel cis-regulatory elements in murine hematopoiesis

BackgroundThe differentiation of hematopoietic stem and progenitor cells (HSPCs) to terminally differentiated immune cells is accompanied by large-scale remodeling of the DNA methylation landscape. While significant insights into the molecular mechanisms of hematopoietic tissue regeneration were derived from mouse models, profiling of DNA methylation changes has been hampered by high cost or low resolution using the methods available. This problem has been overcome by the recent development of the Infinium Mouse Methylation BeadChip (MMBC) array, facilitating methylation profiling of the mouse genome at single CpG resolution at affordable cost. ResultsWe extended the RnBeads package to provide a computational framework for the analysis of MMBC data. This framework was applied to a newly generated MMBC reference map of mouse hematopoiesis encompassing nine different cell types. The analysis of dynamically regulated CpG sites showed progressive and unidirectional DNA methylation changes from HSPCs to differentiated hematopoietic cells and allowed the identification of lineage- and cell type-specific DNA methylation programs. Comparison to previously published catalogues of cis-regulatory elements (CREs) revealed 12,856 novel putative CREs which were dynamically regulated by DNA methylation (mdCREs). These mdCREs were predominantly associated with patterns of cell type-specific DNA hypomethylation and could be identified as epigenetic control regions regulating the expression of key hematopoietic genes during differentiation. ConclusionsWe established a publicly available analysis pipeline for MMBC datasets and provide a DNA methylation atlas of mouse hematopoiesis. This resource allowed us to identify novel putative CREs involved in hematopoiesis and will serve as a platform to study epigenetic regulation of normal and malignant hematopoiesis.

genomics↗

bioRxiv2021

Loss of Usp22 enhances histone H2B monoubiquitination and stimulates intracellular and systemic interferon immunity

Interferons protect from virus infections by inducing hundreds of interferon-stimulated genes (ISG) which orchestrate anti-viral adaptive and innate immunity. Upon viral infection or type I interferon (IFN) stimulation of cell lines, a histone modification, monoubiquitinated histone 2B (H2Bub1), increases at ISG loci, raising the possibility that a specific chromatin state can broadly stimulate IFN immunity in vivo. Here we show that, in the absence of virus infection or elevated interferon levels, mice lacking the relevant deubiquitinase, Usp22, in immune cells have elevated H2Bub1 levels. Hypermonoubiquitinated H2B is physically associated with dozens of ISG loci, and expression of large numbers of ISG is upregulated. This epigenetic state promotes intracellular and systemic immune phenotypes akin to adaptive and innate interferon immunity, and thereby identifies Usp22 as a negative regulator of interferon immunity.

immunology↗

bioRxiv2020

Hematopoietic stem cells fail to regenerate following inflammatory challenge.

Hematopoietic stem cells (HSCs) are canonically defined by their capacity to maintain the HSC pool via self-renewal divisions. However, accumulating evidence suggests that HSC function is instead preserved by sustaining long-term quiescence. Here, we study the kinetics of HSC recovery in mice, following an inflammatory challenge that induces HSCs to exit dormancy. Repeated inflammatory challenge resulted in a progressive depletion of functional HSCs, with no sign of later recovery. Underlying this observation, label retention experiments demonstrated that self-renewal divisions were absent or extremely rare during challenge, as well as during any subsequent recovery period. While depletion of functional HSCs held no immediate consequences, young mice exposed to inflammatory challenge developed blood and bone marrow hypocellularity in old age, similar to elderly humans. The progressive, irreversible attrition of HSC function demonstrates that discreet instances of inflammatory stress can have an irreversible and therefore cumulative impact on HSC function, even when separated by several months. These findings have important implications for our understanding of the role of inflammation as a mediator of dysfunctional tissue maintenance and regeneration during ageing.

cell biology↗

bioRxiv2020

AmpliconDesign - An interactive web server for the design of high-throughput targeted DNA methylation assays

Targeted analysis of DNA methylation patterns based on bisulfite-treated genomic DNA (BT-DNA) is considered as a gold-standard for epigenetic biomarker development. Existing software tools facilitate primer design, primer quality control or visualization of primer localization. However, high-throughput design of primers for BT-DNA amplification is hampered by limits in throughput and functionality of existing tools, requiring users to repeatedly perform specific tasks manually. Consequently, the design of PCR primers for BT-DNA remains a tedious and time-consuming process. To bridge this gap, we developed AmpliconDesign, a webserver providing a scalable and user-friendly platform for the design and analysis of targeted DNA methylation studies based on BT-DNA, e.g. deep amplicon bisulfite sequencing (ampBS-seq), EpiTYPER MassArray, or pyrosequencing. Core functionality of the web server includes high-throughput primer design and binding site validation based on in silico bisulfite-converted DNA sequences, prediction of fragmentation patterns for EpiTYPER MassArray, an interactive quality control as well as a streamlined analysis workflow for ampBS-seq. Availability and ImplementationThe AmpliconDesign webserver is freely available online at: https://amplicondesign.dkfz.de/. AmpliconDesign has been implemented using the R Shiny framework (Chang et al., 2018). The source code is publicly available under the GNU General Public License v3.0 (https://github.com/MaxSchoenung/AmpliconDesign). ContactDaniel B. Lipka (d.lipka@dkfz.de) & Maximilian Schonung (m.schoenung@dkfz.de)

bioinformatics↗

bioRxiv2020

Methylome-based cell-of-origin modeling (Methyl-COOM) identifies aberrant expression of immune regulatory molecules in CLL

BackgroundIn cancer, normal epigenetic patterns are disturbed and contribute to gene expression changes, disease onset and progression. The cancer epigenome is composed of the epigenetic patterns present in the tumor-initiating cell at the time of transformation, and the tumor-specific epigenetic alterations that are acquired during tumor initiation and progression. The precise dissection of these two components of the tumor epigenome will facilitate a better understanding of the biological mechanisms underlying malignant transformation. Chronic lymphocytic leukemia (CLL) originates from differentiating B cells, which undergo extensive epigenetic programming. This poses the challenge to precisely determine the epigenomic ground-state of the cell-of-origin in order to identify CLL-specific epigenetic aberrations. MethodsWe developed a linear regression model, methylome-based cell-of-origin modeling (Methyl-COOM), to map the cell-of-origin for individual CLL patients based on the continuum of epigenomic changes during normal B cell differentiation. ResultsMethyl-COOM accurately maps the cell-of-origin of CLL and identifies CLL-specific aberrant DNA methylation events that are not confounded by physiologic epigenetic B cell programming. Furthermore, Methyl-COOM unmasks abnormal action of transcription factors, altered super-enhancer activities, and aberrant transcript expression in CLL. Among the aberrantly regulated transcripts were many genes that have previously been implicated in T cell biology. Flow cytometry analysis of these markers confirmed their aberrant expression on malignant B cells at the protein level. ConclusionsMethyl-COOM analysis of CLL identified disease-specific aberrant gene regulation. The aberrantly expressed genes identified in this study might play a role in immune-evasion in CLL and might serve as novel targets for immunotherapy approaches. In summary, we propose a novel framework for in silico modeling of reference DNA methylomes and for the identification of cancer-specific epigenetic changes, a concept that can be broadly applied to other human malignancies.

cancer biology↗