Florian Halbritter

• PhD
• Principal Investigator at Children's Cancer Research Institute

Langerhans cell histiocytosis (LCH) is a rare neoplasm predominantly affecting children. It occupies a hybrid position between cancers and inflammatory diseases, which makes it an attractive model for studying cancer development. To explore the molecular mechanisms underlying the pathophysiology of LCH and its characteristic clinical heterogeneity,...

INTRODUCTION Embryonic development and tissue homeostasis depend on cooperation between specialized cell types. Resident macrophages are professional phagocytes that survey their surroundings; eliminate unfit cells, microorganisms, and metabolic waste; and produce a large range of bioactive molecules and growth factors. Resident macrophages also se...

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here...

Single-cell RNA sequencing (scRNA-seq) has become a standard approach to investigate molecular differences between cell states. Comparisons of bioinformatics methods for the count matrix transformation (normalization) and differential expression (DE) analysis of these data have already highlighted recommendations for effective between-sample compar...

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analys...

A prime goal of regenerative medicine is to replace dysfunctional cells in the body. To design protocols for producing target cells in the laboratory, one may need to consider exponentially large combinations of culture components. Here, we investigated the potential of iteratively approximating the target phenotype by quantifying the distance betw...

Ewing sarcoma (EwS) is a highly aggressive pediatric cancer driven by the EWS::FLI1 (EF) fusion oncoprotein. Emerging evidence suggests that variations in EF expression levels may contribute to tumor cell plasticity, promoting treatment resistance and relapse. Both tumor intrinsic and extrinsic influences such as microenvironmental and therapy rela...

Ewing sarcoma (ES) is a rare but aggressive cancer, with survival rates of only about 30% in metastatic cases. Its rarity and severity highlight the need for focused research, yet progress is limited by scarce patient samples, few animal models, and minimal preclinical testing platforms. Conventional 2D cell cultures often fail to replicate primary...

Metastasis is the primary cause of mortality in pediatric cancer, with the lung being a common site for secondary tumor growth. A major challenge in treatment is the lack of models that accurately replicate the interactions between tumors and the lung metastatic niche observed in patients. This limitation hinders the investigation of the molecular...

The mechanisms underlying tumor cell plasticity driving drug resistance and disease progression remain poorly understood. In Ewing sarcoma (EwS), variations in EWS::FLI1 (EF) activity have been associated with epithelial-mesenchymal plasticity (EMP). Using degron technology, we titrated endogenous EF in an EwS cell line and linked phenotypic states...

Ewing sarcoma (EwS) is an aggressive cancer of adolescents in need of effective treatment. Insulin-like growth factor (IGF)-1 is an autocrine growth factor for EwS, but only 10% of patients respond to IGF-1 receptor (IGF-1R) blockade. Although EwS is presumed to originate from mesenchymal progenitors during bone development, targeting of the EwS dr...

We present a single-arm, phase II, neoadjuvant trial with the oncolytic virus talimogene laherparepvec (T-VEC) in 18 patients with difficult-to-resect cutaneous basal cell carcinomas. The primary end point, defined as the proportion of patients, who after six cycles of T-VEC (13 weeks), become resectable without the need for plastic reconstructive...

The term cancer immunoediting describes the dual role by which the immune system can suppress and promote tumour growth and is divided into three phases: elimination, equilibrium and escape. The role of NK cells has mainly been attributed to the elimination phase. Here we show that NK cells play a role in all three phases of cancer immunoediting. E...

Langerhans cell histiocytosis (LCH) is a clonal hematopoietic disorder defined by tumorous lesions containing CD1a+/CD207+ cells. Two severe complications of LCH are systemic hyperinflammation and progressive neurodegeneration. The scarcity of primary samples and lack of appropriate models limit our mechanistic understanding of LCH pathogenesis and...

Secondary lymphoid organs (SLOs) provide the confined microenvironment required for stromal cells to interact with immune cells to initiate adaptive immune responses resulting in B cell differentiation. Here, we studied three patients from two families with functional hyposplenism, absence of tonsils, and complete lymph node aplasia, leading to rec...

Ewing sarcoma is a highly aggressive pediatric cancer driven by the EWS::FLI1 (EF) fusion oncogene. Evidence suggests that EF expression levels dictate tumor cell plasticity contributing to treatment resistance and relapse. Our study aims to elucidate the transcriptional programs and phenotypes associated with distinct EF thresholds. To dynamically...

Ewing sarcoma (EwS) is an aggressive cancer of adolescents in need of effective treatments. Insulin like growth factor (IGF) 1 was previously reported an autocrine growth factor for EwS, but only 10% of patients responded to IGF-1 receptor blockade. Although presumed to originate from mesenchymal progenitors during bone development, targeting of th...

Immune cells need to sustain a state of constant alertness over a lifetime. Yet, little is known about the regulatory processes that control the fluent and fragile balance that is called homeostasis. Here we demonstrate that JAK-STAT signaling, beyond its role in immune responses, is a major regulator of immune cell homeostasis. We investigated JAK...

Ewing sarcoma (ES) is an aggressive pediatric bone tumor in adolescents primarily driven by the expression of the fusion oncogene EWS::FLI1. Although the etiology of ES remains controversial, human mesenchymal stem cells (hMSCs) are considered to be the likely cell of origin. This is not only due to their ability to tolerate ectopic EWS::FLI1 expre...

Metastasis remains the main cause of death in pediatric cancer patients with the lung being one of the most common affected organs. Our inability to cure these patients is largely due to insufficient knowledge about tumor and metastatic niche plasticity, while existing models are not fully effective in mirroring the response seen in clinical settin...

Although KDM5C is one of the most frequently mutated genes in X-linked intellectual disability¹, the exact mechanisms that lead to cognitive impairment remain unknown. Here we use human patient-derived induced pluripotent stem cells and Kdm5c knockout mice to conduct cellular, transcriptomic, chromatin and behavioural studies. KDM5C is identified a...

The term cancer immunoediting describes the dual role by which the immune system can both suppress and promote tumour growth and is divided into three phases: elimination, equilibrium and escape. While the importance of CD8+ T cells in all three phases of cancer immunoediting is well-established, the role of NK cells has mainly been attributed to t...

The NFAT family of transcription factors plays central roles in adaptive immunity in murine models, however, their contribution to human immune homeostasis remains poorly defined. In a multigenerational pedigree, we identified three patients carrying germline biallelic missense variants in NFATC1, presenting with recurrent infections, hypogammaglob...

Early childhood malignancies are driven by sparse genetic aberrations in oncogenes that often co-occur with large copy number variants (CNVs). The combination of these mutations is thought to transform developmentally pliant embryonic cells to initiate tumorigenesis. However, the mechanistic interactions between CNVs, oncogenes, and differentiation...

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xe...

Neutrophils are evolutionarily conserved innate defense cells implicated in diverse pathological processes. Zebrafish models have contributed substantially to our understanding of neutrophil functions, but similarities to human neutrophil maturation have not been characterized limiting applicability to study human disease. We generated transgenic z...

Early childhood tumours are thought to arise from transformed embryonic cells, which often carry large copy number variants (CNVs). However, it remains unclear how CNVs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ human embryonic stem cell (hESC) differentiation to assess the effects of chromosome 17q/1q g...

The neural crest (NC) is an important multipotent embryonic cell population and its impaired specification leads to various developmental defects, often in an anteroposterior (A-P) axial level-specific manner. The mechanisms underlying the correct A-P regionalisation of human NC cells remain elusive. Recent studies have indicated that trunk NC cell...

Ewing sarcoma (EwS) is driven by the EWS-FLI1 (EF) oncoprotein and is assumed to originate from mesenchymal progenitors during bone development. Attempts to generate a EwS genetic mouse model by targeting EF expression to the mesenchymal lineage so far failed to result in tumorigenesis. Here, we investigated the influence of bone niche-derived fact...

Osteosarcoma (OS) and Ewing sarcoma (ES) are the most common bone cancers in children. They are rare cancers and thus difficult to study due to scarcity of patient material, large genomic instability and a wide histological heterogeneity (in OS) or a lack of satisfactory transgenic animal model and availability of preclinical tests (in ES). There i...

Ewing sarcoma (EwS), an aggressive bone and soft tissue cancer predominantly occurring during adolescence, is driven by the chimeric EWS-FLI1 oncogene. It is assumed that a permissive cellular context or potentially additional complementing mutations are necessary for the EWS-FLI1 fusion to exert its full oncogenic effect. Prior efforts to generate...

Introduction: Neuroblastoma is the most common solid tumor in infants and arises from progenitor cells during sympathoadrenal development. While the majority of primary tumor cells resembles adrenergic neurons, more undifferentiated mesenchymal or neural-crest cell-like phenotypes have been found, especially in pretreated and high-risk cases. In mo...

Helios, encoded by IKZF2, is a member of the Ikaros family of transcription factors with pivotal roles in T-follicular helper, NK- and T-regulatory cell physiology. Somatic IKZF2 mutations are frequently found in lymphoid malignancies. Although germline mutations in IKZF1 and IKZF3, encoding Ikaros and Aiolos, have recently been identified in patie...

Helios, a member of the Ikaros family of transcription factors, is predominantly expressed in developing thymocytes, activated T cells, and regulatory T cells (Tregs). Studies in mice have emphasized its role in maintenance of Treg immunosuppressive functions by stabilizing Foxp3 expression and silencing the Il2 locus. However, its contribution to...

Langerhans cell histiocytosis (LCH) is a neoplasm marked by the accumulation of CD1A+CD207+ cells. It is most commonly driven by a somatic, activating mutation in the BRAF serine-threonine kinase (BRAFV600E). Multisystem disease with risk-organ involvement requires myelotoxic chemotherapy, making BRAF-inhibitors an attractive treatment option. Here...

Background In early-stage mycosis fungoides (MF), the most common primary cutaneous T-cell lymphoma, limited skin involvement with patches and plaques is associated with a favorable prognosis. Nevertheless, approximately 20–30% of cases progress to tumors or erythroderma, resulting in poor outcome. At present, factors contributing to this switch fr...

Background In early-stage mycosis fungoides (MF), the most common primary cutaneous T-cell lymphoma, limited skin involvement with patches and plaques is associated with a favorable prognosis. Nevertheless, approximately 20–30% of cases progress to tumors or erythroderma, resulting in poor outcome. At present, factors contributing to this switch fr...

The neural crest (NC) is an important multipotent embryonic cell population and its impaired specification leads to various developmental defects, often in an anteroposterior (A-P) axial level-specific manner. The mechanisms underlying the correct A-P regionalisation of human NC cells remain elusive. Recent studies have indicated that trunk NC cell...

While the bone marrow attracts tumor cells in many solid cancers leading to poor outcome in affected patients, comprehensive analyses of bone marrow metastases have not been performed on a single-cell level. We here set out to capture tumor heterogeneity and unravel microenvironmental changes in neuroblastoma, a solid cancer with bone marrow involv...

The transcription factors STAT5A and STAT5B are critical in hematopoiesis and leukemia. They are widely believed to have redundant functions but we describe a unique role for STAT5B in driving the self-renewal of hematopoietic and leukemic stem cells (HSCs/LSCs). We find STAT5B to be specifically activated in HSCs and LSCs, where it induces many ge...

In mammalian genomes, differentially methylated regions (DMRs) and histone marks including trimethylation of histone 3 lysine 27 (H3K27me3) at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. However, neither parent-of-origin-specific transcription nor imprints have been comprehensively mapped at the blasto...

Ewing sarcoma is a pediatric bone and soft tissue cancer for which new therapies to improve disease outcome and to reduce adverse effects of current standard treatments are urgently needed. To identify new and effective drugs, phenotypic drug screening has proven to be a powerful method and a cancer model ideally suited for this approach is the lar...

CD8 ⁺ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8 ⁺ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I bin...

Many genes are regulated by multiple enhancers that often simultaneously activate their target gene. However, how individual enhancers collaborate to activate transcription is not well understood. Here, we dissect the functions and interdependencies of five enhancer elements that together activate Fgf5 expression during exit from naive murine pluri...

In mammals, chromatin marks at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. This control is thought predominantly to involve parent-specific differentially methylated regions (DMR) in genomic DNA. However, neither parent-of-origin-specific transcription nor DMRs have been comprehensively mapped. We here...

Bone marrow commonly serves as a metastatic niche for disseminated tumor cells (DTCs) of solid cancers in patients with unfavorable clinical outcome. Single-cell assessment of bone marrow metastases is essential to decipher the entire spectrum of tumor heterogeneity in these cancers, however, has previously not been performed. Here we used multi-ep...

Langerhans cell histiocytosis (LCH) is a rare neoplasm predominantly affecting children. It occupies a characteristic hybrid position between cancer and inflammatory disease, which makes it an attractive model for studying cancer development. Constitutive activation of the ERK signaling pathway is a common feature of LCH, but its pathogenesis remai...

The evolution of human diets led to preferences toward polyunsaturated fatty acid (PUFA) content with ‘Western’ diets enriched in ω-6 PUFAs. Mounting evidence points to ω-6 PUFA excess limiting metabolic and cognitive processes that define longevity in humans. When chosen during pregnancy, ω-6 PUFA-enriched ‘Western’ diets can reprogram maternal bo...

Langerhans Cell Histiocytosis (LCH) is a neoplastic disease characterized by the accumulation of CD1a⁺CD207⁺ cells of unknown origin. Constitutive activation of the ERK signaling pathway is a common feature of LCH, but the pathogenesis of this disease remains unclear. Here we present a comprehensive analysis of the composition of LCH lesions that p...

Langerhans Cell Histiocytosis (LCH) is a neoplastic disease characterized by the accumulation of CD1a⁺CD207⁺ cells of unknown origin. Constitutive activation of the ERK signaling pathway is a common feature of LCH, but the pathogenesis of this disease remains unclear. Here we present a comprehensive analysis of the composition of LCH lesions that p...

The International Stem Cell Initiative compared several commonly used approaches to assess human pluripotent stem cells (PSC). PluriTest predicts pluripotency through bioinformatic analysis of the transcriptomes of undifferentiated cells, whereas, embryoid body (EB) formation in vitro and teratoma formation in vivo provide direct tests of different...

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Self-renewal of embryonic stem cells (ESCs) cultured in LIF/fetal calf serum (FCS) is incomplete with some cells initiating differentiation. While this is reflected in heterogeneous expression of naive pluripotency transcription factors (TFs), the link between TF heterogeneity and differentiation is not fully understood. Here, we purify ESCs with d...

Table S2. Enhancer Activities and Modules, Related to Figures 2, 3, 4, 5, 6, and 7

Data S1. Genomic Coordinates (BED Files) of ChIP-Seq Peaks, ChIP-STARR-Seq Enhancer with Activity Level, and Super-Enhancers Called in This Study, Related to Figures 1, 2, 5, and 7

Table S5. Oligonucleotides Used in This Study, Related to Figures 1, 2, 3, 4, 5, 6, and 7

Enhancers are genetic elements that regulate spatiotemporal gene expression. Enhancer function requires transcription factor (TF) binding and correlates with histone modifications. However, the extent to which TF binding and histone modifications functionally define active enhancers remains unclear. Here, we combine chromatin immunoprecipitation wi...

Table S1. Full List of Genes Expressed in Wild-Type and cKO HSCs Isolated from Untreated or 5-FU-Treated Mice, Related to Figures 2 and S2

Hematopoietic stem cells (HSCs) sustain hematopoiesis throughout life. HSCs exit dormancy to restore hemostasis in response to stressful events, such as acute blood loss, and must return to a quiescent state to prevent their exhaustion and resulting bone marrow failure. HSC activation is driven in part through the phosphatidylinositol 3-kinase (PI3...

Deletion of Sox2 from mouse embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here we show that Sox2 can be deleted from EpiSCs with impunity. This is d...

Microarray gene expression data described in Figure 1.

List of the cell lines used in this study with their genotype and additional transgenes if present.

List of the PCR primers and sgRNAs used in this study.

Deletion of Sox2 from mouse embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here, we show that Sox2 can be deleted from EpiSCs with impunity. This is...

Deletion of Sox2 from embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here we show that Sox2 can be deleted from EpiSCs with impunity. This is due to...

STAT5B is often mutated in hematopoietic malignancies. The most frequent STAT5B mutation, Asp642His (N642H), has been found in over 90 leukemia and lymphoma patients. Here, we used the Vav1 promoter to generate transgenic mouse models that expressed either human STAT5B or STAT5BN642H in the hematopoietic compartment. While STAT5B-expressing mice la...

Enhancers are genetic elements that regulate spatiotemporal gene expression. Enhancer function requires transcription factor (TF) binding and correlates with histone modifications. However, the extent to which TF binding and histone modifications can functionally define active enhancers remains unclear. Here we combine chromatin immunoprecipitation...

Table listing the annotation data and sequencing details for 639 DNA methylation profiles based on the μWGBS/scWGBS protocol and for 13 gene expression profiles based on the Smart-seq2 protocol.

Table S2. Differentially Methylated Regions Between Hematopoietic Cell Types and Lineages, Related to Figures 2 and 3 Table listing all regulatory regions from the BLUEPRINT Regulatory Build that were differentially methylated in at least one pairwise comparison between HSCs and MPPs derived from four different sources or between the myeloid and l...

Table S3. Enrichment Analysis for Differentially Methylated Regions and Cell-Type Signature Regions, Related to Figures 2, 3, and 7 Region set enrichment analysis for differentially methylated regions (Table S2) and cell type signature regions (Table S4) calculated using the LOLA software tool and the LOLA Core database.

Table S4. Signature Regions Identified by the Cell-Type Classifier, Related to Figure 7 Table listing all regulatory regions from the BLUEPRINT Regulatory Build that contributed to the cell type classifier trained on 319 stem/progenitor samples (all 10-cell, 50-cell, and 1,000-cell pools) from peripheral blood, together with the average DNA methyl...

Table S5. Classifier-Based Similarity among the Stem and Progenitor Cell Types, Related to Figure 7 Class probabilities for each stem/progenitor sample by ten classifiers trained on datasets that excluded all samples of one specific cell type (“leave-one-class-out classifiers”).

Hematopoietic stem cells give rise to all blood cells in a differentiation process that involves widespread epigenome remodeling. Here we present genome-wide reference maps of the associated DNA methylation dynamics. We used a meta-epigenomic approach that combines DNA methylation profiles across many small pools of cells and performed single-cell...

Hematopoietic stem cells give rise to all blood cells in a differentiation process that involves widespread epigenome remodeling. Here we present genome-wide reference maps of the associated DNA methylation dynamics. We used a meta-epigenomic approach that combines DNA methylation profiles across many small pools of cells and performed single-cell...

DNA methylation patterns are altered in numerous diseases and often correlate with clinically relevant information such as disease subtypes, prognosis and drug response. With suitable assays and after validation in large cohorts, such associations can be exploited for clinical diagnostics and personalized treatment decisions. Here we describe the r...

Tissue resident macrophages develop during embryogenesis, are maintained independently of hematopoietic stem cells during adulthood, and display tissue-specific functions and phenotypes. To understand the genetic program that drives macrophage differentiation from distinct progenitors and their tissue-specific identity, we performed a systematic sp...