Biology subjects

Fan, A. C.

Publications and source records attributed to Fan, A. C..

7 recordsLinked to original sources

bioRxiv2026

Single cell multi-omics enables high-resolution identification and functionalpurification of human acute myeloid leukemia stem cells

In human acute myeloid leukemia (AML), a sub-population of leukemia stem cells (LSCs) drive disease initiation, therapeutic resistance, and relapse. However, the lack of reliable markers to distinguish LSCs from bulk leukemia cells has impeded progress in studying LSC pathogenesis and developing meaningful LSC-specific diagnostics and therapeutics. Existing LSC gene signatures, derived from bulk populations, cannot definitively identify LSCs at single-cell resolution. To address this, we analyzed large patient cohorts with bulk gene expression data and single-cell multi-omic assays to identify a prognostic gene signature that is specifically enriched in a clinically adverse AML sub-population. Using this signature, we defined and prospectively isolated CD34+CD90-CLL1-CD69+CD53- immunophenotypic LSCs that are significantly enriched for LSC content based on limiting dilution xenotransplantation assays. Our findings demonstrate the power of single-cell multi-omics to precisely identify a clinically relevant LSC population and establish a clear framework for future translational research in AML. Key PointsO_LISingle cell multi-omics identifies human AML LSCs at high resolution. C_LIO_LIHOPX and SOCS2 co-expression (hrLSC2) defines a prognostic gene signature in de novo acute myeloid leukemia. C_LIO_LIhrLSC2 marks an AML subpopulation (iLSCs) with a distinct immunophenotype. C_LIO_LIiLSCs can be purified using flow cytometry and are significantly enriched for LSCs. C_LI

cancer biology↗

bioRxiv2025

Submicron-Sampling of Living Cells by Macrophages

SUMMARY PARAGRAPHAn effective immune system must sample and appreciate healthy-self identity to prevent autoimmunity and to contrast to pathogenic insults1-3. Self-proteins are presented to T cells in the thymus during immune cell development2,3, and must be presented throughout the body to both maintain regulatory T cell populations4-6 and provide a tonic signal to maintain conventional T cells over time7-9. The ready observations of continuous apoptosis in some organs together with the ingestion of that material by myeloid populations has led to a conventional understanding of ongoing cell-death as a major source of self-antigens10, complemented in some situations by uptake of free-floating cell-derived vesicles. Here, we used a series of companion imaging and vesicular labeling technologies to reveal an alternate process undertaken by macrophages that results in non-destructive and direct sampling of living cells. The process requires cell-cell contact, does not require caspase activation, and takes place via a trogocytosis-like stretching of the target cell into the macrophage, leading to the generation of submicron-sized vesicles containing cytoplasm. Using a high-dimensional flow-based method for labeling vesicles ingested under this versus other conditions, we find that live-sampled material is distinctly processed, is poorly subject to fusion with lysosomes, and produces ensuing differential effects on the presentation of those to CD4 versus CD8 T cells. Disrupting this trafficking by redirecting antigen to the lysosome significantly reduced the associated macrophage-mediated priming of CD8 T cells. This demonstrates an important and substantial sampling of living cells by the immune system, with clear consequences for maintaining the border of immunity.

immunology↗

bioRxiv2024

DNMT3AR882H Is Not Required for Disease Maintenance in Primary Human AML, but Is Associated With Increased Leukemia Stem Cell Frequency

Genetic mutations are being thoroughly mapped in human cancers, yet a fundamental question in cancer biology is whether such mutations are functionally required for cancer initiation, maintenance of established cancer, or both. Here, we study this question in the context of human acute myeloid leukemia (AML), where DNMT3AR882 missense mutations often arise early, in pre-leukemic clonal hematopoiesis, and corrupt the DNA methylation landscape to initiate leukemia. We developed CRISPR-based methods to directly correct DNMT3AR882 mutations in leukemic cells obtained from patients. Surprisingly, DNMT3AR882 mutations were largely dispensable for disease maintenance. Replacing DNMT3AR882 mutants with wild-type DNMT3A did not impair the ability of AML cells to engraft in vivo, and minimally altered DNA methylation. Taken together, DNMT3AR882 mutations are initially necessary for AML initiation, but are largely dispensable for disease maintenance. The notion that initiating oncogenes differ from those that maintain cancer has important implications for cancer evolution and therapy. STATEMENT OF SIGNIFICANCEUnderstanding which driver mutations are required for cancer initiation, maintenance, or both phases remains poorly understood. Here, we uncover that highly prevalent pre-leukemic DNMT3A mutations are only required during disease initiation, but become dispensable after leukemic transformation, uncovering the context-specific role of this driver mutation with important therapeutic implications.

cancer biology↗

bioRxiv2024

Combined Nanodrops Imaging and Ultrasound Localization Microscopy for Detecting Intracerebral Hemorrhage

ObjectiveAdvanced imaging methods are crucial for understanding stroke mechanisms and discovering effective treatments to reduce bleeding and enhance recovery. In preclinical in vivo stroke imaging, MRI, CT, and optical imaging are commonly used to evaluate stroke outcomes in rodent models. However, MRI and CT have limited spatial resolution for rodent brains, and optical imaging is hindered by limited imaging depth of penetration. Here we introduce a novel contrast-enhanced ultrasound imaging method to overcome these challenges and characterize intracerebral hemorrhage with unique insights. MethodsWe combined microbubble (MB)-based ultrasound localization microscopy (ULM) and nanodrop (ND)- based vessel leakage imaging to achieve simultaneous microvascular imaging and hemorrhage detection. ULM maps brain-wide cerebral vasculature with high spatial resolution and identifies microvascular impairments around hemorrhagic areas. NDs are sub-micron liquid-core particles which can extravasate due to blood-brain barrier (BBB) breakdown, serving as positive contrast agents to detect hemorrhage sites. ResultsOur findings demonstrate that NDs could effectively accumulate in the hemorrhagic site and reveal the location of the bleeding areas upon activation by focused ultrasound beams. ULM further reveals the microvascular damage manifested in the form of reduced vascularity and decreased blood flow velocity across areas affected by the hemorrhagic stroke. ConclusionThe results demonstrate that sequential ULM combined with ND imaging is a useful imaging tool for basic in vivo research in stroke with rodent models where brain-wide detection of active bleeding and microvascular impairment are essential.

bioengineering↗

bioRxiv2024

Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR)

Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge and serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies of substrate-dependent target engagement remain sparse. Herein, we describe a strategy for the discovery of substrate-dependent ligand binding. Using proteome integral solubility alteration (PISA) assays, we show that simple biochemical additives can enable detection of RNA-protein-small molecule complexes in native cell lysates. We apply our approach to rocaglates, molecules that specifically clamp RNA to eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), and potentially other members of the DEAD-box (DDX) helicase family. To identify unexpected interactions, we used a target class-specific thermal window and compared ATP analog and RNA base dependencies for key rocaglate-DDX interactions. We report and validate novel DDX targets of high-profile rocaglates - including the clinical candidate Zotatifin - using limited proteolysis-mass spectrometry and fluorescence polarization (FP) experiments. We also provide structural insight into divergent DDX3X affinities between synthetic rocaglates. Taken together, our study provides a model for screening uncompetitive inhibitors using a chemical proteomics approach and uncovers actionable DDX clamping targets, clearing a path towards characterization of novel molecular clamps and associated RNA helicases.

biochemistry↗

bioRxiv2023

Diverse and Abundant Viruses Exploit Conjugative Plasmids

Phages exert profound evolutionary pressure on bacteria by interacting with receptors on the cell surface to initiate infection. While the majority of phages use chromosomally-encoded cell surface structures as receptors, plasmid-dependent phages exploit plasmid-encoded conjugation proteins, making their host range dependent on horizontal transfer of the plasmid. Despite their unique biology and biotechnological significance, only a small number of plasmid-dependent phages have been characterized. Here we systematically search for new plasmid-dependent phages targeting IncP and IncF plasmids using a targeted discovery platform, and find that they are common and abundant in wastewater, and largely unexplored in terms of their genetic diversity. Plasmid-dependent phages are enriched in non-canonical types of phages, and all but one of the 64 phages we isolated were non-tailed, and members of the lipid-containing tectiviruses, ssDNA filamentous phages or ssRNA phages. We show that plasmid-dependent tectiviruses exhibit profound differences in their host range which is associated with variation in the phage holin protein. Despite their relatively high abundance in wastewater, plasmid-dependent tectiviruses are missed by metaviromic analyses, underscoring the continued importance of culture-based phage discovery. Finally, we identify a tailed phage dependent on the IncF plasmid, and find related structural genes in phages that use the orthogonal type 4 pilus as a receptor, highlighting the promiscuous use of these distinct contractile structures by multiple groups of phages. Taken together, these results indicate plasmid-dependent phages play an under-appreciated evolutionary role in constraining horizontal gene transfer via conjugative plasmids.

microbiology↗

bioRxiv2022

A genome-wide atlas of recurrent repeat expansions in human cancer

Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases. However, repeat expansions are often not explored beyond neurological and neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs (STRs), a phenomenon termed microsatellite instability (MSI); however larger repeat expansions have not been systematically analyzed in cancer. Here, we identified TR expansions in 2,622 cancer genomes, spanning 29 cancer types. In 7 cancer types, we found 160 recurrent repeat expansions (rREs); most of these (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with an enrichment near candidate cis-regulatory elements, suggesting a role in gene regulation. One rRE located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, targeting cells harboring this rRE with a rationally designed, sequence-specific DNA binder led to a dose-dependent decrease in cell proliferation. Overall, our results demonstrate that rREs are an important but unexplored source of genetic variation in human cancers, and we provide a comprehensive catalog for further study.

genomics↗