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Halene, S.

Publications and source records attributed to Halene, S..

17 recordsLinked to original sources
bioRxiv2026

Biomarker-Targeted O6-Guanine Alkylation Potentiates ATR Inhibitor Response in De Novo and Relapsed/Refractory AML

Acute myeloid leukemia (AML) remains limited by high relapse rates and a scarcity of biomarker-directed therapies, underscoring the need to identify actionable vulnerabilities and mechanisms of therapeutic resistance. O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that directly reverses mutagenic O6-alkylguanine lesions, is epigenetically silenced in multiple cancers. Although MGMT silencing is predictive of temozolomide (TMZ) response in glioblastoma with significant survival advantage, prior clinical trials of TMZ in AML have shown only modest responses. Therefore, the prevalence and therapeutic relevance of MGMT silencing, as well as genetic factors that modify its therapeutic response in AML, remain to be comprehensively investigated. Here, we first profiled MGMT status across a diverse cohort of 23 de novo and 16 relapsed/refractory (R/R) primary AMLs using an integrated analysis of MGMT mRNA expression, promoter methylation, and protein expression. We found that approximately 25-30% of AMLs harbor MGMT silencing, in contrast to consistent MGMT expression in healthy CD34+ hematopoietic stem and progenitor cells. We further identified frequent loss of mismatch repair (MMR) proteins in these AML cohorts, a known resistance mechanism against TMZ in glioblastoma. Using CRISPR knockout screening and genetically engineered AML models, we found that MMR loss also drives both upfront and acquired TMZ resistance in MGMT-silenced AML. These findings identify MMR deficiency as an important and previously underappreciated genetic contributor to the modest responses observed in prior trials of TMZ in AML. To therapeutically exploit MGMT silencing while bypassing genetic determinants of resistance, including MMR deficiency, we evaluated clinically and preclinically characterized MGMT-dependent DNA-alkylating agents and identified a fluoroethylating analog of TMZ, N3-(2-fluoroethyl) imidazotetrazine (KL50), with pronounced and selective activity in MGMT-silenced AML. KL50 retained antileukemic activity irrespective of MMR status, significantly prolonging survival in humanized MISTRG6 mice harboring primary AML patient-derived xenografts. Mechanistically, KL50 induced DNA damage through the time-dependent formation of interstrand DNA crosslinks, bypassing MMR dependence and triggering a replication stress response dominated by ATR signaling. Pharmacologic ATR inhibition synergized with KL50, producing marked antileukemic activity and significantly extending survival across AML models without compromising hematologic safety. Together, these findings establish MGMT silencing as a prevalent and therapeutically actionable biomarker in AML, define MMR status as a key determinant of TMZ response in AML but not KL50 sensitivity, and provide a translational rationale for combining low-dose O6-fluoroethylating imidazotetrazines with ATR inhibitors to target both de novo and R/R AML across diverse genetic backgrounds.

cancer biology↗
bioRxiv2026

Human Lymph Node Cellular Senescence Atlas Reveals Age-Dependent Alteration in Germinal Center B Cell Function and Niches

Immunosenescence, the age-associated decline in immune function, is a key feature of human aging. In human lymphoid organs, however, the specific immune cell populations that acquire senescence-associated phenotypes during aging and how they influence the surrounding tissue microenvironment remain poorly understood. A spatially resolved map of these senescence-associated immune states in human lymphoid tissues could help clarify their relationship with aging and their potential contributions to the progressive decline of immune function. Here, we integrated single-cell and spatial multi-omics to systematically characterize age-related senescence in human lymph nodes (LNs). Single-cell transcriptomics of lymphoid tissues from donors aged 18 to 100 years old identified 34 immune and stromal cell types and revealed age-associated upregulation of senescence signatures in specific populations. Spatial proteomic profiling of 99 LN sections from 51 donors (18-86 years) using high-plex immunofluorescence ([~]20 million cells) mapped senescence markers (p16, p21, HMGB1, -H2AX) at single-cell resolution, revealing diverse senescent-like cell types ("senotypes") and a stepwise shift from extrafollicular to germinal center (GC) localization with age. Notably, we observed focal clonal-like senescence in GC B cells in older donor LNs. Spatial transcriptomics, epigenomics, and metabolic imaging of selected samples further elucidate the multi-omics signatures and underlying mechanisms of functional impairment, metabolic remodeling, and distinct regulatory programs in senescent-like GC B cells. This study presents a comprehensive spatial atlas of senescence-associated immune states in human lymph nodes, revealing cell-type-specific and spatial heterogeneity that may contribute to immunosenescence and the decline of immune function during aging.

immunology↗
bioRxiv2025

Unique epigenomic signatures identify biologically significant subtypes of MDS and predict response to azacitidine

Myelodysplastic syndromes (MDS) are characterized by aberrant DNA methylation, and mutations in epigenetic modifiers are frequently found in these patients. Although DNA methyltransferase inhibitors (DNMTi) are used to treat MDS, response variability remains a challenge in the clinic, with limited predictive markers. Through comprehensive genomic, epigenomic, and transcriptomic analyses, we have gained valuable insights into the intricate interplay between genetic and epigenetic alterations in MDS. We describe aberrantly hyper and hypomethylated regions in MDS, extending beyond promoter regions and affecting long-distance regulatory elements. Using these aberrant DNA methylation patterns, we classified MDS patients into epigenetic subtypes correlated with known molecular drivers. This epigenetic classification includes a novel group of patients characterized only by their shared DNA methylation profile and lacking any genetic drivers. Furthermore, we identified a robust DNA methylation signature capable of distinguishing DNMTi responders from non-responders prior to receiving treatment. Leveraging these DMRs, we developed robust classifiers capable of predictive response to DNMTi by integrating DNA methylation, gene expression, mutations, and laboratory parameters. Our findings highlight the potential of epigenetic-based classifiers for personalized treatment approaches for MDS patients. Key PointsDNA methylation patterns define biologically meaningful MDS subtypes and uncover a new group lacking known mutations. A methylation-based signature at diagnosis predicts azacitidine response, supporting its use in guiding personalized MDS therapy.

cancer biology↗
bioRxiv2025

RBM15-MKL1 fusion protein promotes leukemia via m6A methylation and WNT pathway activation

Acute megakaryoblastic leukemia driven by the RBM15-MKL1 fusion protein (RM-AMKL) is the only known recurrent mutation involving the N6-methyladenosine (m6A) writer complex. Dysregulation of m6A modification affects RNA fate and is linked to oncogenesis. Inhibition of m6A deposition via inhibition of the METTL3 writer protein has anti-tumour activity, but the mechanism underlying its efficacy and cancer specificity remains unclear. We treated murine RM-AMKL cells with a novel METTL3 inhibitor, STM3675, and showed apoptosis in vitro and prolonged survival of mice transplanted with RM-AMKL, implicating m6A as an essential component of AMKL and identifying Wnt signalling as a key driver of leukemogenesis. To elucidate the mechanism by which m6A contributes to leukemogenesis we employed a multi-omic approach, combining transcriptome-wide assessment of RNA binding, methylation and turnover. We show for the first time that RM retains the RNA-binding and m6A-modifiying functions of its RBM15 component, while also selectively regulating distinct mRNA targets, particularly genes involved in Wnt signalling including Frizzled. Frizzled genes are upregulated by RM and downregulated in RM-AMKL cells in response to METTL3 inhibition, providing an m6A-dependent explanation for their upregulation. Direct Frizzled knockdown reduced RM-AMKL growth, which was partially rescued by treatment with a {beta}-catenin agonist, underscoring a functional role of Wnt signalling in RM-AMKL. Human AMKLs show elevated Wnt pathway and Frizzled gene expression, highlighting the relevance of our work. Together, our findings reveal that RM-specific m6A modifications and activation of Wnt signalling are critical drivers of RM-AMKL, highlighting these pathways as potential therapeutic targets. Key PointsO_LIRM retains functional abilities of RBM15 and additionally interacts with Wnt-related transcripts to increase expression of Fzd proteins. C_LIO_LIThe METTL3 writer complex and WNT signalling pathways are essential for RM-driven leukemia. C_LI

cancer biology↗
bioRxiv2024

An in vivo screen identifies NAT10 as a master regulator of brain metastasis

Metastasis is the major cause of cancer-related deaths. Emerging evidence has shown that epigenetic regulation plays a fundamental role in cancer metastasis. To better understand the epigenetic regulation of metastasis, we conducted an in vivo shRNA screen for vulnerabilities of brain metastasis and identified N-acetyltransferase 10 (NAT10) as a driver of brain metastasis. Knockdown of NAT10 significantly restrains cancer cell proliferation and migration in vitro, and tumor growth and brain metastasis in vivo. Structure-function analysis of NAT10 showed that its poorly characterized RNA helicase domain is critical for breast cancer cell growth in vitro, while its N-acetyltransferase domain is essential for primary tumor growth and brain metastasis in vivo. Integrative transcriptomic and proteomic analyses revealed key downstream effectors of NAT10, including PHGDH and PSAT1, two catalyzing enzymes for serine biosynthesis implicated in brain metastasis, and HSPA5, known to promote metastasis. We found that distant metastases of breast cancer, especially brain metastases express higher levels of NAT10, PHGDH, PSAT1, and HSPA5. Silencing PHGDH/PSAT1 or HSPA5 in metastatic breast cancer cells inhibits their ability to grow in the serine/glycine-limited condition or migrate, respectively, phenocopying the effects of NAT10 depletion. Moreover, NAT10 promotes the expression of PHGDH, PSAT1, and HSPA5 in its RNA helicase-dependent manner. These findings establish NAT10 as a master regulator of brain metastasis and shed light on the biological functions of its RNA helicase domain, nominating NAT10 as a target for treating metastatic diseases.

cancer biology↗
bioRxiv2024

Interpreting single-cell messages in normal and aberrant hematopoiesis with the Cell Marker Accordion

Single-cell technologies offer a unique opportunity to explore cellular heterogeneity in health and disease. However, reliable identification of cell types and states represents a bottleneck. Available databases and analysis tools employ dissimilar markers, leading to inconsistent annotations and poor interpretability. Furthermore, current tools focus mostly on physiological cell types, limiting their applicability to disease. We developed the Cell Marker Accordion, a user-friendly platform providing automatic annotation and unmatched biological interpretation of single-cell populations, based on consistency weighted markers. We validated our approach on multiple single-cell and spatial datasets from different human and murine tissues, improving annotation accuracy in all cases. Moreover, we show that the Cell Marker Accordion can identify disease-critical cells and pathological processes, extracting potential biomarkers in a wide variety of disease contexts. The breadth of these applications elevates the Cell Marker Accordion as a fast, flexible, faithful and standardized tool to annotate and interpret single-cell and spatial populations in studying physiology and disease.

bioinformatics↗
bioRxiv2023

Impact of Memory T Cells on SARS-COV-2 Vaccine Response in Hematopoietic Stem Cell Transplant

During the COVID-19 pandemic, hematopoietic stem cell transplant (HSCT) recipients faced an elevated mortality rate from SARS-CoV-2 infection, ranging between 10-40%. The SARS-CoV-2 mRNA vaccines are important tools in preventing severe disease, yet their efficacy in the post-transplant setting remains unclear, especially in patients subjected to myeloablative chemotherapy and immunosuppression. We evaluated the humoral and adaptive immune responses to the SARS-CoV-2 mRNA vaccination series in 42 HSCT recipients and 5 healthy controls. Peripheral blood mononuclear nuclear cells and serum were prospectively collected before and after each dose of the SARS-CoV-2 vaccine. Post-vaccination responses were assessed by measuring anti-spike IgG and nucleocapsid titers, and antigen specific T cell activity, before and after vaccination. In order to examine mechanisms behind a lack of response, pre-and post-vaccine samples were selected based on humoral and cellular responses for single-cell RNA sequencing with TCR and BCR sequencing. Our observations revealed that while all participants eventually mounted a humoral response, transplant recipients had defects in memory T cell populations that were associated with an absence of T cell response, some of which could be detected pre-vaccination.

immunology↗
bioRxiv2022

Microfluidic immuno-serology assay revealed a limited diversity of protection against COVID-19 in patients with altered immunity

The immune response to SARS-CoV-2 for patients with altered immunity such as hematologic malignancies and autoimmune disease may differ substantially from that in general population. These patients remain at high risk despite wide-spread adoption of vaccination. It is critical to examine the differences at the systems level between the general population and the patients with altered immunity in terms of immunologic and serological responses to COVID-19 infection and vaccination. Here, we developed a novel microfluidic chip for high-plex immuno-serological assay to simultaneously measure up to 50 plasma or serum samples for up to 50 soluble markers including 35 plasma proteins, 11 anti-spike/RBD IgG antibodies spanning all major variants, and controls. Our assay demonstrated the quintuplicate test in a single run with high throughput, low sample volume input, high reproducibility and high accuracy. It was applied to the measurement of 1,012 blood samples including in-depth analysis of sera from 127 patients and 21 healthy donors over multiple time points, either with acute COVID infection or vaccination. The protein association matrix analysis revealed distinct immune mediator protein modules that exhibited a reduced degree of diversity in protein-protein cooperation in patients with hematologic malignancies and patients with autoimmune disorders receiving B cell depletion therapy. Serological analysis identified that COVID infected patients with hematologic malignancies display impaired anti-RBD antibody response despite high level of anti-spike IgG, which could be associated with limited clonotype diversity and functional deficiency in B cells and was further confirmed by single-cell BCR and transcriptome sequencing. These findings underscore the importance to individualize immunization strategy for these high-risk patients and provide an informative tool to monitor their responses at the systems level.

bioengineering↗
bioRxiv2022

Spatial-CITE-seq: spatially resolved high-plex protein and whole transcriptome co-mapping

We present spatial-CITE-seq for high-plex protein and whole transcriptome co-mapping, which was firstly demonstrated for profiling 189 proteins and transcriptome in multiple mouse tissue types. It was then applied to human tissues to measure 273 proteins and transcriptome that revealed spatially distinct germinal center reaction in tonsil and early immune activation in skin at the COVID-19 mRNA vaccine injection site. Spatial-CITE-seq may find a range of applications in biomedical research.

genomics↗
bioRxiv2022

Human neutrophil development and functionality are enabled in a humanized mouse model

Mice with a functional human immune system serve as an invaluable tool to study the development and function of human immune system in vivo. A major technological limitation of all current humanized mouse models is the lack of mature and functional human neutrophils in circulation and tissues. To overcome this, we generated a humanized mouse model named MISTRGGR, in which the mouse granulocyte colony stimulating factor (G-CSF) was replaced with human G-CSF and the mouse G-CSF receptor gene was deleted (G-CSFR) in existing MISTRG mice. By targeting the GCSF cytokine-receptor axis, we dramatically improved the reconstitution of mature circulating and tissue-infiltrating human neutrophils in MISTRGGR mice. Moreover, these functional human neutrophils in MISTRGGR are recruited upon inflammatory and infectious challenges and help reduce bacterial burden. MISTRGGR mice represent a unique mouse model that finally permits the study of human neutrophils in health and disease. Key PointsO_LITargeting the GCSF cytokine-receptor axis dramatically improves circulating and tissue-infiltrating human neutrophils in MISTRGGR mice. C_LIO_LIHuman neutrophils generated in MISTRGGR mice are functional and are able to respond robustly to inflammatory and infectious stimuli. C_LI

immunology↗
bioRxiv2021

Viral replication in human macrophages enhances an inflammatory cascade and interferon driven chronic COVID-19 in humanized mice.

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA, and sustained interferon (IFN) response all of which are recapitulated and required for pathology in the SARS-CoV-2 infected MISTRG6-hACE2 humanized mouse model of COVID-19 with a human immune system1-20. Blocking either viral replication with Remdesivir21-23 or the downstream IFN stimulated cascade with anti-IFNAR2 in vivo in the chronic stages of disease attenuated the overactive immune-inflammatory response, especially inflammatory macrophages. Here, we show SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release IL-1 and IL-18 and undergo pyroptosis thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and its accompanying inflammatory response is necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Remarkably, this same blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 by production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.

immunology↗
bioRxiv2021

Pre-mRNA Splicing Factor U2AF2 Recognizes Distinct Conformations of Nucleotide Variants at the Center of the pre-mRNA Splice Site Signal

The essential pre-mRNA splicing factor U2AF2 (also called U2AF65) identifies polypyrimidine (Py) tract signals of nascent transcripts, despite length and sequence variations. Previous studies have shown that the U2AF2 RNA recognition motifs (RRM1 and RRM2) preferentially bind uridine-rich RNAs. Nonetheless, the specificity of the RRM1/RRM2 interface for the central Py tract nucleotide has yet to be investigated. We addressed this question by determining crystal structures of U2AF2 bound to a cytidine, guanosine, or adenosine at the central position of the Py tract, and compared U2AF2-bound uridine structures. Local movements of the RNA site accommodated the different nucleotides, whereas the polypeptide backbone remained similar among the structures. Accordingly, molecular dynamics simulations revealed flexible conformations of the central, U2AF2-bound nucleotide. The RNA binding affinities and splicing efficiencies of structure-guided mutants demonstrated that U2AF2 tolerates nucleotide substitutions at the central position of the Py tract. Moreover, enhanced crosslinking and immunoprecipitation of endogenous U2AF2 in human erythroleukemia cells showed uridine-sensitive binding sites with lower sequence conservation at the central nucleotide positions of otherwise uridine-rich, U2AF2-bound splice sites. Altogether, these results highlight the importance of RNA flexibility for protein recognition and take a step towards relating splice site motifs to pre-mRNA splicing efficiencies.

biophysics↗
bioRxiv2021

Spatial-ATAC-seq: spatially resolved chromatin accessibility profiling of tissues at genome scale and cellular level

Cellular function in tissue is dependent upon the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping, but it remains elusive to capture spatial epigenetic information of tissue at cellular level and genome scale. Here we report on spatial-ATAC-seq: spatially resolved chromatin accessibility profiling of tissue section via next-generation sequencing by combining in situ Tn5 transposition chemistry and microfluidic deterministic barcoding. Spatial chromatin accessibility profiling of mouse embryos delineated tissue region-specific epigenetic landscapes and identified gene regulators implicated in the central nerve system development. Mapping the accessible genome in human tonsil tissue with 20m pixel size revealed spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology takes spatial biology to a new realm by enabling spatially resolved epigenomics to improve our understanding of cell identity, state, and fate decision in relation to epigenetic underpinnings in development and disease.

genomics↗
bioRxiv2021

Multi-omics profiling of U2AF1 mutants dissects pathogenic mechanisms affecting RNA granules in myeloid malignancies

Somatic mutations in splicing factors are of significant interest in myeloid malignancies and other cancers. U2AF1, together with U2AF2, is essential for 3 splice site recognition. U2AF1 mutations result in aberrant splicing, but the molecular mechanism and the full spectrum of consequences on RNA biology have not been fully elucidated to date. We performed multi-omics profiling of in vivo RNA binding, splicing and turnover for U2AF1 S34F and Q157R mutants. We dissected specific binding signals of U2AF1 and U2AF2 and showed that U2AF1 mutations individually alter U2AF1-RNA binding, resulting in defective U2AF2 recruitment. We demonstrated a complex relationship between differential binding and splicing, expanding upon the currently accepted loss-of-binding model. Finally, we observed that U2AF1 mutations increase the formation of stress granules in both cell lines and primary acute myeloid leukemia samples. Our results uncover U2AF1 mutation-dependent pathogenic RNA mechanisms and provide the basis for developing targeted therapeutic strategies.

cancer biology↗
bioRxiv2020

MCL1 dependence across MDS subtypes and dual inhibition of MCL1 and BCL2 in MISTRG6 mice

Treatment for myelodysplastic syndromes (MDS) remains insufficient due to clonal heterogeneity and clinical complexity. Dysregulation of apoptosis is observed across MDS subtypes regardless of mutations and represents an attractive therapeutic opportunity. Venetoclax (VEN), a selective inhibitor of anti-apoptotic protein B-cell lymphoma-2 (BCL2), has yielded impressive responses in older patients with acute myeloid leukemia (AML). BCL2 family anti-apoptotic proteins BCL-XL and induced myeloid cell leukemia 1 (MCL1) are implicated in leukemia survival, and upregulation of MCL1 is seen in VEN-resistant AML and MDS. Here, we determined the in vitro sensitivity of MDS patient samples to selective inhibitors of BCL2, BCL-XL and MCL1. While VEN response positively correlated with increasing blast counts, all MDS subtypes responded to the MCL1 inhibitor, S63845. Treatment with combined VEN+S63845 was synergistic in all MDS subtypes and reduced MDS engraftment in MISTRG6 mice supporting the pursuit of clinical trials with combined BCL2+MCL1 inhibition in MDS.

cancer biology↗
bioRxiv2018

A Highly Efficient and Faithful MDS Patient-Derived Xenotransplantation Model for Pre-Clinical Studies

Comprehensive preclinical studies of Myelodysplastic Syndromes (MDS) have been elusive due to limited ability of MDS stem cells to engraft current immunodeficient murine hosts. We developed a novel MDS patient-derived xenotransplantation model in cytokine-humanized immunodeficient \"MISTRG\" mice that for the first time provides efficient and faithful disease representation across all MDS subtypes. MISTRG MDS patient-derived xenografts (PDX) reproduce patients' dysplastic morphology with multi-lineage representation, including erythro- and megakaryopoiesis. MISTRG MDS-PDX replicate the original sample's genetic complexity and can be propagated via serial transplantation. MISTRG MDS-PDX demonstrate the cytotoxic and differentiation potential of targeted therapeutics providing superior readouts of drug mechanism of action and therapeutic efficacy. Physiologic humanization of the hematopoietic stem cell niche proves critical to MDS stem cell propagation and function in vivo. The MISTRG MDS-PDX model opens novel avenues of research and long-awaited opportunities in MDS research.

cancer biology↗